Virulence genes, proteins, and their use

ABSTRACT

A series of genes from  Pseudomonas aeruginosa  and  Klebsiella  are shown to encode products that are implicated in virulence. The identification of theses genes therefore allows attenuated microorganisms to be produced. Furthermore, the genes or their encoded products can be used to identify antimicrobial drugs, diagnostic methods for the identification of a pathogen-associated disease, and in the manufacture of vaccines.

FIELD OF THE INVENTION

This invention relates to virulence genes and proteins, and their use. More particularly, it relates to genes and proteins/peptides obtained from gram-negative bacteria, and their use in therapy and in screening for drugs.

BACKGROUND OF THE INVENTION

According to health care experts, infectious diseases caused by microbes are responsible for more deaths worldwide than any other single cause. The current estimate of the annual cost of medical care for treating infectious diseases in the United States alone is about $120 billion. While antibiotic treatment is effective for many microbial infections, antibiotic resistance among pathogenic bacteria is a growing health concern. Indeed, the American Medical Association has concluded that, “the global increase in resistance to antimicrobial drugs, including the emergence of bacterial strains that are resistant to all available antibacterial agents, has created a public health problem of potentially crisis proportions.”

Pseudomonas and Klebsiella are two genuses of gram-negative bacteria that pose a significant health risk to infected host organisms, in part, due to their resistance to many antibiotics. These bacteria are noted for causing life-threatening infections, particularly in the lung. Cancer and burn patients also commonly suffer serious Pseudomonas infections, as do certain other individuals with immune system deficiencies. While Klebsiella sp. is responsible for many types of infections, outside of a medical setting, the most common infection caused by Klebsiella bacteria is pneumonia.

There is a need in the art for new antimicrobial therapeutic strategies.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery of 46 genes, when mutated lower the virulence of a gram-negative bacterium, and can be used in new antimicrobial therapeutic strategies. The invention provides attenuated bacterial mutants that are derived from pathogenic strains. These attenuated bacterial stains have a mutation in a VIRX gene identified herein as VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14, VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR43, VIR44, VIR45, and VIR46; and show reduced inhibition of Dictyostelium amoeba growth when compared to the growth observed in the presence of an isogenic bacterial strain. The term, “pathogenic,” as used herein, is defined as an agent's ability to cause disease, damage or harm to a host organism. The term, “attenuated,” as used herein, means an organism made less virulent relative to an isogenic pathogenic organism. The term, “mutant,” as used herein, an organism carrying a specific mutation of a gene that is expressed in the organism's phenotype. A mutation may be insertional inactivation or deletion of a gene. It is preferred that the mutation be an insertional inactivation of a gene.

The invention also provides attenuated bacterial mutants that are derived from pathogenic gram-negative bacterial strains. These attenuated gram-negative bacterial strains have a mutation in a VIRX gene identified herein as VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14, VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR43, VIR44, VIR45, and VIR46; and show reduced inhibition of Dictyostelium amoeba growth when compared to the growth observed in the presence of an isogenic bacterial strain. A mutation may be insertional inactivation or deletion of a gene. It is preferred that the mutation be an insertional inactivation of a gene. It is also preferred that the attenuated gram-negative bacterial mutant be derived from a Pseudomonas or Klebiella spp. It is more preferred that the attenuated gram-negative bacterial mutant is a strain of P. aeruginosa or K. pneumoniae.

The invention additionally provides for a VIRX gene that may be part of an operon. The term, “operon,” as used herein, is a unit of bacterial gene expression and regulation comprising several genes, usually with complementary functions. Insertion in a gene in an operon typically interferes with the function of this gene and of other genes located downstream or upstream in the operon. The function attributed to a gene refers to its function and/or that of any gene located downstream or upstream in the same operon. Accordingly, the invention also provides for a bacterial strain comprising an operon encoding a gene selected from the group consisting of VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14, VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR44, VIR45, and VIR46, wherein the bacterial strain includes a mutation that reduces expression of the VIRX gene relative to an isogenic bacterial strain lacking the mutation. In one embodiment, the the mutation reduces inhibition of Dictyostelium amoeba growth when compared to the growth of Dictyostelium amoeba in the presence of an isogenic bacterial strain lacking the mutation.

The invention provides for one or more of the following attenuated Pseudomonas mutant strains: MUT1; MUT2; MUT3; MUT4; MUT5; MUT6; MUT7; MUT8; MUT9; MUT10; MUT11; MUT12; MUT13; MUT14; MUT15: MUT16; MUT17; MUT18; and MUT19. The invention also provides for one or more of the following attenuated Klebsiella mutant strains: MUT20; MUT21; MUT22; MUT23; MUT24; MUT25; MUT26; MUT27; MUT28; MUT29; MUT30; MUT31; MUT32; MUT33; MUT34; MUT35; MUT36; MUT37; MUT38; MUT39; MUT40; MUT41; MUT42; MUT43; MUT44; MUT45; and MUT46.

The invention additionally provides a method for identifying an antimicrobial drug, wherein a candidate composition is contacted with at least one polypeptide encoded by a gene selected from the group consisting of VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14, VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR43, VIR44, VIR45 and VIR46. The biological activity of polypeptide in the presence of the candidate composition is compared with the biological activity of the polypeptide in the absence of the candidate composition. Alteration of the biological activity of the polypeptide indicates that the candidate composition is an antimicrobial drug. In some embodiments, the candidate composition contains at least two molecules. The candidate composition can contain at least one molecule less than about 500 Daltons or at least one molecule greater than about 500 Daltons. The candidate composition can be, e.g., an immunoglobulin, polysaccharide, lipid, nucleic acid, or combination thereof.

The invention additionally provides a method for identifying an antimicrobial drug, wherein a candidate composition is contacted with at least one polynucleotide encoded by a gene selected from the group consisting of VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14, VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR43, VIR44, VIR45, and VIR46. The expression of the polynucleotide in the presence of the candidate composition is compared with the expression of the polynucleotide in the absence of the candidate composition. Alteration of the expression of the polynucleotide indicates that the candidate composition is an antimicrobial drug. In some embodiments, the candidate composition contains at least two molecules. The candidate composition can contain at least one molecule less than about 500 Daltons or at least one molecule greater than about 500 Daltons. The candidate composition can be a polypeptide, polysaccharide, lipid, nucleic acid, e.g., ribonucleic acid, or combination thereof. In a preferred embodiment, the ribonucleic acid of the candidate composition is a small interfering ribonucleic acid.

The invention additionally provides a method for determining the degree of virulence of a pathogen present in a subject, comprising:

-   -   (a) measuring the level of expression of at least one         polypeptide encoded by a gene selected from the group consisting         of VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10,         VIR11, VIR12, VIR13, VIR14, VIR15, VIR16, VIR17, VIR18, VIR19,         VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28,         VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37,         VIR38, VIR39, VIR40, VIR41, VIR42, VIR43, VIR44, VIR45, and         VIR46, in a sample from the first subject; and     -   (b) comparing the amount of the polypeptide in the sample of         step (a) to the amount of the polypeptide present in a control         sample from a second subject known not to have the presence of         the pathogen, wherein an alteration in the expression level of         the polypeptide in the first subject as compared to the control         sample indicates the degree of virulence of the pathogen.

In a preferred embodiment, the subject is a mammal. It is more preferred that the subject is a human.

The invention also provides a method for determining the degree of virulence of a pathogen present in a subject, comprising:

(a) measuring the level of expression of at least one polynucleotide encoded by a gene selected from the group consisting of VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14, VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR5, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR44, VIR45, and VIR46, in a sample from the first subject; and

(b) comparing the amount of the polynucleotide in the sample of step (a) to the amount of the polynucleotide present in a control sample from a second subject known not to have the presence of the pathogen, wherein an alteration in the expression level of the polypeptide in the first subject as compared to the control sample indicates the degree of virulence of the pathogen.

In a preferred embodiment, the subject is a mammal. It is more preferred that the subject is a human.

The invention additionally provides attenuated bacterial strains that can be used as vaccines and as vectors for foreign antigens and for foreign DNA. These attenuated bacterial strains are useful for the preparation of vaccines effective against diseases associated with the corresponding bacterial strains. In a preferred embodiment, the attenuated bacterial strains are derived from Pseudomonas or Klebsiella spp.

The invention additionally provides attenuated bacterial strains that can be used as vectors for foreign genes cloned from other pathogens that will be expressed into proteins, and will raise protective immune responses against the pathogens from which they are derived. In a preferred embodiment, the attenuated bacterial strains used as the vectors are derived from Pseudomonas or Klebsiella spp.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery of 46 genes when mutated lower the virulence of a gram-negative bacterium. Nineteen of these virulence genes were identified in P. aeruginosa PT894, while the remaining 27 genes were derived from mutagenesis of Klebsiella. These bacterial mutants have attenuated virulence relative to isogenic bacterial strains and are designated “MUX.” Provided herein are virulence genes affected in each novel, attenuated MUTX strain, as well as the nucleotides and polypeptides encoded thereby. The sequences encoded by the affected genes are collectively referred to as “VIRX nucleic acids” or “VIRX polynucleotides” and the corresponding encoded polypeptides are referred to as “VIRX polypeptides” or “VIRX proteins.” Unless indicated otherwise, “VIRX” is meant to refer to any of the novel sequences disclosed herein.

The peptides and genes of the invention are useful for the preparation of therapeutic agents to treat infection because they attenuate the virulence of the wild-type pathogen. Therapy can be preventative or therapeutic. A subject receiving therapy can be, e.g., a human, a non-human primate (such as an ape, gorilla, or chimpanzee), cow, horse, pig, sheep, dog, cat, or rodent (including mouse or rat).

I. Identification of Pseudomonas and Klebsiella Genes Encoding Virulence Factors

Genes encoding virulence factors (e.g., pathogens or toxins) to a host organism were identified by comparing the growth of Dictyostelium discoideum, in the presence and absence of test mutants of Pseudomonas and Klebsiella with an identifiable genetic alteration as detailed in International Application PCT/IB02/03277, filed Jun. 7, 2002. Dictyostelium amoebae feed phagocytically upon bacteria such as K. pneumoniae. When Dictyostelium cells are plated with K. pneumoniae bacteria, each amoeba creates a plaque in the bacterial lawn in the region where bacteria have been phagocytosed. Addition of pathogenic bacteria, e.g., P. aeruginosa strain PT894 to the lawn of K. pneumoniae bacteria, inhibits the growth of the amoebae.

Pseudomonas test mutants were made by transposon insertion according to known methods in the art and tested for virulence in a Dictyostelium growth assay (see, PCT/IB02/03277, filed Jun. 7, 2002). Klebsiella mutants were also made by transposon insertion according to known methods in the art and tested for virulence in a Dictyostelium growth assay (see, PCT/IB02/03277, filed Jun. 7, 2002) using the PHG1a mutant Dictyostelium strain (Cornillon et al., J. Biol. Chem., 275(44): 34287-92, 2000), a strain which was found to be particularly sensitive to virulent bacteria. Specifically, the Klebsiella mutants were obtained by standard bacteria electroporation technique using the plasposon pNKBOR (Genbank accession number: AF310136) and selected on solid LB medium containing 50 μg/ml kanamycin (Rossignol et al., Res. Microbiol., 152(5): 481-5, 2001). Other mutagenesis methods known in the art, e.g., ultraviolet radiation exposure, treatment with intercalating agent or transducing phage, may also be used to generate mutants. Mutations yielding reduced virulence were identified where the growth of the Dictyostelium test host organism exposed to the mutant pathogen was greater than the Dictyostelium test host organism exposed to wild-type pathogen. Specific genetic mutations in pathogens displaying reduced virulence were subsequently identified and characterized by techniques well known in the art. Identification of specific gene mutations in Klebsiella mutants was performed by plasmid rescue and cloning of the genomic DNA at the insertion site mutant using the BglII or ApaI restriction enzyme according to (Rossignol et al., Res. Microbiol., 152(5): 481-5, 2001). Identification of specific gene mutations in Pseudomonas mutants was performed by subcloning the transposon and surrounding bacteria genomic DNA into an acceptor plamid. DNA sequencing was performed on amplified rescued plasmids, in order to identify the insertion site of the transposon. Rat mortality assays such as that described by Join-Lambert et al., Antimicrob. Agents Chemother., 45(2): 571-6, 2001, can be used to corroborate attenuated virulence activity in a mammalian host.

The 19 Pseudomonas attenuated MUTX organisms harboring the VIRX genes are summarized below in Table 1. TABLE 1 STRAIN AFFECTED VIRULENCE GENE(S) REFERENCE MUT1 anthranilate phosphoribosyltransferase Essar et al, J. Bacteriol., 172: 853-66, (trpD; PA0650) 1990; Essar et al., J. Bacteriol., 172: 867- 83, 1990. MUT2 ATP sulfurylase small subunit Leyh et al., J. Biol. Chem., 263: 2409- (CysD; PA4443) 16, 1988; Hummerjohann et al., Microbiology, 144 (Pt 5): 1375-86, 1998 MUT3 CysQ (PA5175) Peng and Verma, J. Biol. Chem., 270: 29105-10, 1995; Neuwald et al., J. Bacteriol., 174: 415-25, 1992. MUT4 D-amino acid dehydrogenase, small subunit Lobacka et al., J. Bacteriol., 176: 1500-10, (dadA; PA5304) 1994. MUT5 imidazoleglycerol-phosphate synthase, cyclase Fani et al., Mol. Gen. Genet., 216: 224-9, subunit (hisF1; PA5140) 1989; Fani et al., Mol. Gen. Genet., 216: 224-9, 1989. MUT6 N-acetyl-γ-glutamyl-phosphate reductase Smith et al., Gene, 49: 53-60. 1986. (ArgC; PAO 0662) MUT7 Dihydrolipoamide acetyltransferase (AceF; Rae et al., J. Bacteriol., 179: 3561-71, pyruvate dehydrogenase complex component 1997. E2; PA5016) MUT8 NADH dehydrogenase I chain H Weidner et al., J. Mol. Biol., 5: 233: 109- (nuoH; PA2643) 22, 1993; Weidner et al., J. Mol. Biol., 233: 109-22, 1993. MUT9 pyoverdine synthetase D Rombel et al., Mol. Gen. Genet., 246: 519- (PvdD; PA2399) 28, 1995; Merriman et al., J. Bacteriol., 177: 252-8, 1995. MUT10 RND multidrug efflux transporter MexD Poole et al., Mol. Microbiol., 21:713-24, (mexD; PA4598) 1996; Poole et al., Mol. Microbiol., 21: 713-24, 1996. MUT11 PA3721 Stover et al., Nature, 406: 959-964, 2000. MUT12 PA0596 Tan et al., Proc. Natl. Acad. Sci. USA, 96: 2408-13, 1999. MUT13 PA5265 Stover et al., Nature, 406: 959-964, 2000. MUT14 pyochelin biosynthetic protein pchC Serino et al., Mol. Gen. Genet., 249: (PA4229) 217-28, 1995; Serino et al., J. Bactiol., 179: 248-57, 1997 MUT15 dihydroaeruginoic acid synthetase Reimmann et al., Microbiology, 144: (pchE; PA4226) 3135-48, 1998. MUT16 Pyochelin synthetase Reimmann et al., Microbiology, 144: (pchF; PA4225) 3135-48, 1998. MUT17 ATP-binding component of the ABC Featherston et al., Mol. Microbiol., transporter 32(2): 289-99, 1999; Reimmann et al., J. (pchH; PA4223) Bacteriol., 183: 813-20, 2001. MUT18 ATP-binding component of the ABC Reimmann et al., J. Bacteriol., 183: 813-20, transporter (pchI; PA4222) 2001. MUT19 putative O-antigen biosynthesis gene cluster Rocchetta et al., Microbiol. Mol. Biol. Rev. 63: 523-53, 1999.

The 27 Klebsiella attenuated MUTX organisms harboring the VIRX genes disclosed in the present invention and assigned a new role in virulence are summarized below in Table 2. TABLE 2 STRAIN AFFECTED VIRULENCE GENE(S) MUT20 hypothetical transcriptional regulator in met G-dld intergenic region MUT21 β-cystathionase MUT22 ribosome binding factor A MUT23 aspartokinase/homoserine dehydrogenase MUT24 cystathionine γ-synthase MUT25 Phophoribosylformylglycinamidine synthase MUT26 homoserine transsuccinylase MUT27 3′-phosphoadenosine 5′-phosphosulfate reductase MUT28 Sfi protein MUT29 transcriptional activator protein LysR MUT30 TrpD MUT31 N-acetylglucosamine-6-phosphate deacetylase MUT32 WaaQ MUT33 2-Isopropylmalate synthase MUT34 histidinol dehydrogenase MUT35 UDP-galactopyranose mutase MUT36 O-antigen export system permease protein rfba MUT37 uridyltransferase MUT38 pyridoxine phosphate biosynthetic protein PdxJ-PdxA MUT39 triose phosphate isomerase MUT40 aldehyde dehydrogenase MUT41 galactosyl transferase MUT42 siroheme synthetase MUT43 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase MUT44 glucose-6-phosphate isomerase MUT45 DNA methylase MUT46 putative inner membrane protein II. Attenuated Bacterial Mutants

A. Attenuated Pseudomonas aeruginosa Mutants

MUT1

A Pseudomonas bacterial mutant (MUT1) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding anthranilate phosphoribosyltransferase (PA0650). This gene encodes the VIR1 nucleic acid (SEQ ID NO:1) shown in Table 3A. TABLE 3A VIR1 Nucleotide Sequence (SEQ ID NO:1) ATGGATATCAAGGGAGCCCTCAATCGCATCGTCAACCAGCTCGACCTGACCACCGAGGAAATGCAGG CGGTCATGCGCCAGATCATGACCGGGCAGTGCACCGACGCGCAGATCGGCGCCTTCCTGATGGGCAT GCGGATGAAGAGCGAAACCATCGACGAGATCGTCGGCGCGGTGGCGGTGATGCGCGAACTGGCCGAC GGCGTGCAGTTGCCTACGCTGAAGCATGTGGTCGACGTGGTCGGCACCGGCGGCGATGGCGCGAACA TCTTCAACGTGTCCTCGGCGGCGTCCTTCGTGGTCGCCGCCGCTGGCGGCAAGGTCGCCAAACACGG TAACCGCGCGGTCTCCGGCAAGAGCGGCAGCGCCGACTTGCTGGAAGCCGCCGGCATCTACCTGGAG CTGACCTCCGAACAGGTGGCGCGTTGCATCGACACCGTCGGCGTCGGGTTCATGTTCGCCCAGGTCC ACCACAAGGCGATGAACTACGCCGCCGGTCCGCGCCGCGAGCTGGGCTTGCGGACTCTGTTCAACAT GCTTGGCCCACTGACCAACCCGGCGGGACTGAGGCACCAGGTGGTCGGGGTGTTCACCCAGGAACTG TGCAAGCCGCTGGCTGAAGTGCTCAAGCGTCTCGGCAGCGAGCATGTGCTGGTGGTGCATTCGCGCG ACGGGCTGGACGAGTTCAGTCTGGCCGCGGCGACCCACATTGCCGAGTTGAAGGACGGCGAGGTACG CGAGTACGAAGTGCGTCCCGAGGACTTCGGGATCAAGAGCCAGACCCTGATGGGGCTGGAGGTCGAC AGTCCGCAGGCCTCGCTGGAACTGATCCGCGACGCTTTGGGGCGGCGCAAGACCGAGGCTGGGCAGA AGGCCGCCGAGCTGATCGTGATGAATGCCGGCCCGGCACTGTACGCTGCCGATCTGGCGACCAGCCT GCACGAGGGCATTCAACTGGCCCACGATGCCCTGCACACCGGGCTGGCACGGGAGAAGATGGACGAA CTGGTGGCCTTCACCGCCGTTTACAGAGAGGAGAACGCACAGTGA

The VIR1 protein (SEQ ID NO:2) encoded by SEQ ID NO:1 is presented using the one-letter amino acid code in Table 3B. TABLE 3B Encoded VIR1 protein sequence (SEQ ID NO:2) MDIKGALNRIVNQLDLTTEEMQAVMRQIMTGQCTDAQIGAFLMGMRMKSETIDEIVGAVAVMREL ADGVQLPTLKHVVDVVGTGGDGANIFNVSSAASFVVAAAGGKVAKHGNRAVSGKSGSADLLEAAG IYLELTSEQVARCIDTVGVGFMFAQVHHKAMKYAAGPRRELGLRTLFNMLGPLTNPAGVRHQVVG VFTQELCKPLAEVLKRLGSEHVLVVHSRDGLDEFSLAAATHIAELKDGEVREYEVRPEDFGIKSQ TLMGLEVDSPQASLELIRDALGRRKTEAGQKAAELIVMNAGPALYAADLATSLHEGIQLAHDALH TGLAREKMDELVAFTAVYREENAQ

The role of VIR1 in virulence was confirmed using phage to retransduce this mutation into the wild-type PT894 strain where attenuated virulence was again observed in the Dictyostelium growth assay compared to an isogenic bacterial strain.

MUT2

A Pseudomonas bacterial mutant (MUT2) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding the ATP sulfurylase small subunit (CysD; PA4443). This gene encodes the VIR2 nucleic acid (SEQ ID NO:3) shown in Table 4A. TABLE 4A VIR2 Nucleotide Sequence (SEQ ID NO:3) ATGGTCGACAAACTGACGCACCTGAAACAGCTGGAGGCGGAAAGCATCCA CATCATCCGCGAGGTGGCCGCCGAGTTCGATAACCCGGTGATGCTGTACT CGATCGGCAAGGATTCCGCGGTCATGCTGCACCTGGCCCGCAAGGCCTTC TTCCCCGGCAAGCTGCCCTTCCCGGTGATGCACGTGGACACCCGCTGGAA ATTCCAGGAGATGTACAGGTTCCGTGATCGGATGGTCGAGGAAATGGGCC TGGATCTGATCACCCACGTCAACCCGGACGGCGTCGCCCAGGGCATCAAC CCGTTCACCCACGGCAGCGCCAAGCACACCGACGTGATGAAGACCGAGGG ACTCAAGCAGGCCCTGGACAAGTACGGTTTCGACGCTGCCTTCGGCGGTG CGCGCCGCGACGAGGAGAAGTCGCGGGCCAAGGAACGGGTCTATTCGTTC CGCGACAGCAAGCACCGCTGGGACCCGAAGAACCAGCGTCCCGAGCTGTG GAACATCTACAACGGCAAGGTGAAGAAGGGCGAGTCGATCCGCGTCTTCC CGCTGTCCAACTGGACCGAGCTGGACATCTGGCAATACATCTACCTGGAA GGCATCCCGATCGTCCCGCTGTACTTCGCCGCCGAGCGCGAGGTCATCGA GAAGAATGGCACATTGATCATGATCGACGACGAGCGCATCCTCGAGCATC TCTCTGACGAAGAGAAAGCCCGCATCGAGAAGCGCATGGTGCGCTTCCGT ACCCTCGGCTGCTACCCGCTCACCGGCGCGGTCGAGTCCAGCGCCACCAC GCTGCCGGAAATCATCCAGGAAATGCTCCTGACGCGTACTTCCGAACGCC AGGGCCGGGTCATCGACCATGACCAGGCCGGTTCGATGGAAGAAAAGAAA CGTCAGGGCTATTTCTGA

The VIR2 protein (SEQ ID NO:4) encoded by SEQ ID NO:3 is presented using the one-letter amino acid code in Table 4B. TABLE 4B Encoded VIR2 protein sequence (SEQ ID NO:4)hz,1/32 MVDKLTHLKQLEAESIHIIREVAAEFDNPVMLYSIGKDSAVMLHLARKAF FPGKLPFPVMHVDTRWKFQEMYRFRDRMVEEMGLDLITHVNPDGVAQGIN PFTHGSAKHTDVMKTEGLKQALDKYGFDAAFGGARRDEEKSRAKERVYSF RDSKHRWDPKNQRPELWNIYNGKVKKGESIRVFPLSNWTELDIWQYIYLE GIPIVPLYFAAEREVIEKNGTLIMIDDERILEHLSDEEKARIEKRMVRFR TLGCYPLTGAVESSATTLPEIIQEMLLTRTSERQGRVIDHDQAGSMEEKK RQGYF

The role of VIR2 in virulence was confirmed using phage to retransduce this mutation into the wild-type PT894 strain where attenuated virulence was again observed in the Dictyostelium growth assay compared to an isogenic bacterial strain.

MUT3

A Pseudomonas bacterial mutant (MUT3) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding CysQ (PA5175). This gene encodes the VIR3 nucleic acid (SEQ ID NO:5) shown in Table 5A. TABLE 5A VIR3 Nucleotide Sequence (SEQ ID NO:5) ATGAGGCCGGTGCCTTGGGGCGAATTGGTGGCGCTGGTGCGGCGCGCCGGCGAGGCGATCCTGCCGC ACTGGCGCGCCGACCTGGTGGTGCGCTCGAAGGCCGACGAATCGCCGGTGACTGCCGCCGACCTGGC CGCGCACCATATATTGGAGGCGGGATTGCGGGCGCTGGCGCCGGACATTCCGGTGCTTTCCGAAGAG GATTGCGAGATACCGCTGAGCGAGCGCGGCCACTGGCGGCGCTGGTGGCTGGTGGACCCGCTGGACG GCACCAAGGAGTTCATCTCCGGTAGCGAGGAGTTCACCGTCAACGTGGCCCTGGTCGAGGATGGCCG GGTGCTGTTCGGCCTGGTCGGCGTGCCGGTGAGCGGCCGCTGCTACTACGGTGGCGCCGGTCTCGGT GCCTGGCGCGAGGAGGCCGATGGCCGCGCGCAACCGATCAGTGTGCGCCTGGAGCCCGAGGAGGCCT TCACCGTGCTGGCCAGCAAGCGCCATGGCAGCCCGGCCCAGGAGCGCCTGCTGGATGGCTTGAGCGA GCGCTTCGGCGACCTGCGGCGAGCCAGCATCGGCAGTTCGCTGAAGTTCTGCCTGCTGGCCGAGGGC GCTGCCGACTGCTATCCGCGCCTGACGCCAACCTCGCAATGGGACACGGCCGCCGCCCAGGGTGTGC TGGAAGGCGCCGGCGGCGAGGTGCTCGACCTGCATGGTGCGCCATTCACCTACGAGCCGCGCGAGGA TTACCTCAACGGCTCCTTCCTGGCCCTGCCGCGCGCCGCCCAGTGGCGCAGCGAGCTGATCCAACTG GCGCGCGCGCTGCACTGA

The VIR3 protein (SEQ ID NO:6) encoded by SEQ ID NO:5 is presented using, the one-letter amino acid code in Table 5B. TABLE 5B Encoded VIR3 protein sequence (SEQ ID NO:6) MRPVPWGELVALVRRAGEAILPHWRADVVVRDKADESPVTAADLAAHHIL EAGLRALAPDIPVLSEEDCEIPLSERGHWRRWWLVDPLDGTKEFISGSEE FTVNVALVEDGRVLFGLVGVPVSGRCYYGGAGLGAWREEADGRAQPISVR LEPEEAFTVVASKRHGSPAQERLLDGLSERFGDLRRASIGSSLKFCLLAE GAADCYPRLTPTSQWDTAAAQGVLEGAGGEVLDLHGAPFTYEPREDYLNG SFLALPRAAEWRSELIQLARALH MUT4

A Pseudomonas bacterial mutant (MUT4) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding D-amino acid dehydrogenase, small subunit (dadA; PA5304). This gene encodes the VIR4 nucleic acid (SEQ ID NO:7) shown in Table 6A. TABLE 6A VIR4 Nucleotide Sequence (SEQ ID NO:7) ATGCGAGTTCTGGTCCTTGGCAGCGGTGTCATCGGTACCGCCAGTGCGTATTACCTGGCCCGTGCCG GGTTCGAGGTGGTGGTGGTCGACCGTCACCACGGTCCCGCGCTGGAAACCAGCTTCGCCAACGCCGG CCAGGTGTCTCCCGGCTACGCTTCGCCCTGGGCAGCCCCGGGCATTCCCCTGAAGGCCATGAAGTGG CTGCTGGAAAAGCACGCGCCGCTGGCCATCAAGCTCACCTCCGATCCCAGCCAGTACGCCTGGATGC TGCAGATGCTGCGCAACTGCACCGCCGAGCGCTACGCCGTGAACAAGGAGCGCATGGTCCGCCTGTC CGAGTACAGCCGCGATTGCCTCGACGAACTGCGCGCCGAGACCGGCATCGCCTACGAGGGCCGCACC CTCGGCACCACCCAACTGTTCCGCACCCAGGCGCAGCTGGACGCCGCCGGCAAGGACATCGCCGTGC TCGAGCGCTCCGGCGTGCCCTACGAGGTTCTCGACCGCGACGGCATCGCCCGCGTAGAGCCGGCTTT GGCCAAGGTCGCCGACAAGCTGGTCGGCGCCTTGCGCCTGCCCAACGACCAGACCGGCGACTGCCAG CTGTTCACCACCCGCCTGGCGGAAATGGCCAAGGGCCTGGGCGTGGAGTTCCGCTTCGGCCAGAACA TCGAGCGCCTGGACTTCGCCGGCGACCGCATCAACGGCGTGCTGGTCAACGGCGAATTGCTCACCGC CGACCACTACGTGCTGGCCCTGGGCAGCTACTCGCCGCAACTGCTCAAGCCGCTGGGTATCAAGGCT CCGGTCTATCCGCTGAAGGGTTATTCGCTGACCGTGCCGATCACCAACCCGGAGATGGCGCCGACCT CGACCATCCTCGACGAGACCTACAAGGTGGCGATCACCCGCTTCGACCAGCGCATCCGCGTCGGCGG CATGGCGGAAATCGCCGGCTTCGACCTGTCGCTGAACCCGCGCCGCCGCGAGACCCTGGAAATGATC ACCACCGACCTCTATCCCGAGGGCGGCGATATCAGCCAGGCGACCTTCTGGACCGGCCTGCGCCCGG CGACCCCGGATGGCACCCCGATCGTCGGCGCCACCCGCTACCGCAACCTGTTCCTCAATACCGGCCA CGGCACCCTGGGTTGGACCATGGCCTGCGGGTCGGGTCGCTACCTGGCCGACCTGATGGCGAAGAAG CGCCCGCAGATCAGTACCGAAGGCCTGGATATTTCCCGCTACAGCAATTCCCCGGAGAACGCCAAGA ATGCCCATCCAGCGCCAGCACACTAA

The VIR4 protein (SEQ ID NO:8) encoded by SEQ ID NO:7 is presented using the one-letter amino acid code in Table 6B. TABLE 6B Encoded VIR4 protein sequence (SEQ ID NO:8) MRVLVLGSGVIGTASAYYLARAGFEVVVVDRQDGPALETSFANAGQVSPG YASPWAAPGIPLKAMKWLLEKHAPLAIKLTSDPSQYAWMLQMLRNCTART YAVNKERMVRLSEYSRDCLDELRAETGIAYEGRTLGTTQLFRTQAQLDAA GKDIAVLERSGVPYEVLDRDGIARVEPALAKVADKLVGALRLPNDQTGDC QLFTTRLAEMAKGLGVEFRFGQNIERLDFAGDRINGVLVNGELLTADHYV LALGSYSPQLLKPLGIKAPYPLKGYSLTVPITNPEEMAPTSTILDETYKV AITRFDQRIRVGGMAEIAGFDLSLNPRRRETLEMITTDLYPEGGDISQAT FWTGLRPATPDGTPIVGATRYRNLFLNTGHGTLGWTMACGSGRYLADLMA KKRPQISTEGLDISRYSNSPENAKNAHPAPAH

The role of VIR4 in virulence was confirmed using phage to retransduce this mutation into the wild-type PT894 strain where attenuated virulence was again observed in the Dictyostelium growth assay compared to an isogenic bacterial strain.

MUT5

A Pseudomonas bacterial mutant (MUT5) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding imidazoleglycerol-phosphate synthase, cyclase subunit (hisF; PA5140). This gene encodes the VIR5 nucleic acid (SEQ ID NO:9) shown in Table 7A. TABLE 7A VIR5 Nucleotide Sequence (SEQ ID NO:9) ATGGCACTGGCAAAACGCATCATCCCCTGCCTCGACGTGGACAACGGCCGAGTGGTCAAGGGCGTCA AGTTCGAGAACATCCGCGACGCCGGCGACCCGGTCGAGATCGCTCGCCGCTACGACGAGCAGGGTGC CGACGAGATCACCTTCCTCGATATCACCGCCAGCGTCGACGGGCGCGACACCACCCTGCATACCGTC GAGCGCATGGCTAGCCAGGTGTTCATTCCGCTGACCGTGGGCGGCGGCGTACGCAGCGTGCAGGACA TCCGCAACCTGTTGAATGCCGGCGCGGACAAGGTCTCGATCAACACCGCCGCGGTGTTCAACCCCGA GTTCGTCGGTGAGGCCGCCGACCGCTTCGGCTCGCAGTGCATCGTGGTCGCCATCGACGCGAAGAAG GTTTCCGCCCCGGGCGAGGCGCCGCGCTGGGAAATCTTCACCCATGGCGGGCGCAAGCCCACCGGGC TGGATGCCGTGCTCTGGGCGAAGAAGATGGAAGACTTGGGCGCTGGCGAGATTCTCCTGACCAGCAT GGACCAGGACGGCGTGAAGAGCGGTTACGACCTGGGCGTGACCCGCGCCATCAGCGAGGCGGTGAAC GTGCCGGTGATCGCTTCCGGCGGCGTCGGCAACCTGGAGCACCTGGCCGCCGGCATCCTCGAGGGCA AGGCCGACGCGGTGCTCGCGGCGAGCATCTTCCACTTCGGCGAGTACACCGTGCCGGAAGCCAAGGC CTACCTGGCCAGCCGCGGTATCGTGGTGCGCTGA

The VIR5 protein (SEQ ID NO:10) encoded by SEQ ID NO:9 is presented using the one-letter amino acid code in Table 7B. !Encoded VIR5 protein sequence (SEQ ID NO:10) MALAKRIIPCLDVDNGRVVKGVKFENIRDAGDPVEIARRYDEQGADEITF LDITASVDGRDTTLHTVERMASQVFIPLTVGGGVRSVQDIRNLLNAGADK VSINTAAVFNPEFVGEAADRFGSQCIVVAIDAKKVSAPGEAPRWEIFTHG GRKPTGLDAVLWAKKMEDLGAGEILLTSMDQDGVKSGYDLGVTRAISEAV NVPVIASGGVGNLEHLAAGILEGKADAVLAASIFHFGEYTVPEAKAYLAS RGIVVR MUT6

A Pseudomonas bacterial mutant (MUT6) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding N-acetyl-•-glutamyl-phosphate reductase (ArgC; PA0662). This gene encodes the VIR6 nucleic acid (SEQ ID NO:11) shown in Table 8A. TABLE 8A VIR6 Nucleotide Sequence (SEQ ID NO: 11) ATGATCAAGGTCGGCATCGTTGGCGGTACGGGTTATACGGGCGTGGAACTGCTGCGCCTGCTGGCGC AGCATCCGCAGGCCCGGGTGGAAGTGATCACTTCGCGTTCCGAGGCGGGGGTGAAGGTCGCCGACAT GTACCCGAACCTGCGAGGTCATTATGACGACCTGCAGTTCAGCGTGCCGGACGCGCAGCGCCTCGGC GCCTGCCACGTGGTGTTCTTCGCCACGCCGCACGGCGTGGCGCACGCGCTGGCTGGCGAACTGCTGG ACGCCGGGACCCGGGTCATCGATCTGTCCGCTGACTTCCGCCTGGCGGACGCCGAGGAGTGGGCGCG CTGGTACGGCCAGCCGCATGGCGCTCCGGCGCTGCTCGACGAGGCTGTCTACGGCCTGCCGGAAGTG AACCGCGAGAAGATCCGCCAGGCCCGCCTGATCGCCGTGCCGGGCTGCTACCCGACCGCGACCCAGC TGGGCCTGATCCCGCTGCTCGAAGCCGGCCTGGCCGACGCCTCGCGGCTGATCGCCGATTGCAAGTC CGGGGTCAGCGGTGCCGGTCGGGGCGCCAAGGTTGGCTCGCTGTTCTGCGAGGCGGGCGAAAGCATG ATGGCCTACGCGGTCAAAGGGCATCGGCATCTCCCGGAAATCAGCCAGGGCCTGCGTCGGGCCTCCG GCGGCGACGTCGGGCTGACGTTCGTACCGCACCTGACGCCAATGATCCGCGGTATCCATGCAACCCT CTATGCCCATGTCGCGGATCGCTCGGTCGACCTCCAGGCGTTGTTCGAGAAGCGCTACGCCGACGAA CCCTTCGTCGACGTGATGCCGGCCGGCAGCCATCCGGAGACCCGCAGCGTGCGTGGCGCCAATGTCT GCCGAATCGCCGTGCATCGCCCCCAGGGCGGCGACCTGGTGGTGCTGCTGTCGGTGATCGACAACCT GGTCAAGGGCGCCTCGGGTCAGGCGCTCCAGAACATGAACATCCTGTTCGGGCTGGACGAGCGCCTG GGCCTCTCGCATGCGGCCCTGCTCCCCTGA

The VIR6 protein (SEQ ID NO:12) encoded by SEQ ID NO:11 is presented using the one-letter amino acid code in Table 8B. TABLE 8B Encoded VIR6 protein sequence (SEQ ID NO: 12) MIKVGIVGGTGYTGVELLRLLAQHPQARVEVITSRSEAGVKVADMYPNLRGHYDDLQFSVPDAQR LGACDVVFFATPHGVAHALAGELLDAGTRVIDLSADFRLADAEEWARWYGQPHGAPALLDEAVYG LPEVNREKIRQARLIAVPGCYPTATQLGLIPLLEAGLADASRLIADCKSGVSGAGRGAKVGSLFC EAGESMMAYAVKGHRHLPEISQGLRRASGGDVGLTFVPHLTPMIRGIHATLYAHVADRSVDLQAL FEKRYADEPFVDVMPAGSHPETRSVRGANVCRIAVHRPQGGDLVVVLSVIDNLVKGASGQALQNM NILFGLDERLGLSHAALLP

The role of VIR6 in virulence was confirmed using phage to retransduce this mutation into the wild-type PT894 strain where attenuated virulence was again observed in the Dictyostelium growth assay compared to an isogenic bacterial strain.

MUT7

A Pseudomonas bacterial mutant (MM) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding dihydrolipoamide acetyltransferase (AceF; PA5016). This gene encodes the VIR7 nucleic acid (SEQ ID NO:13) is shown in Table 9A. TABLE 9A VIR7 Nucleotide Sequence (SEQ ID NO: 13) GTGAGCGAACTCATTCGCGTACCCGACATCGGCAACGGTGAGGGTGAAGTCATCGAGCTGCTGGTCA AGCCCGGCGACAAGGTCGAGGCCGATCAGAGCCTGCTGACCCTGGAATCCGACAAGGCCAGCATGGA AATCCCCAGTCCCAAGGCCGGGGTAGTGAAAAGCATCAAGGCGAAGGTCGGCGACACCTTGAAAGAA GGTGACGAAATCCTCGAGCTGGAAGTGGAAGGCGGCGAACAGCCTGCCGAAGCCAAGGCCGAGGCAG CGCCCGCCCAACCGGAAGCGCCGAAAGCCGAAGCGCCTGCTCCCGCCCCGAGCGAGAGCAAGCCGGC CGCCCCCGCCGCGGCCAGCGTCCAGGACATCAAGGTCCCGGACATCGGCTCGGCCGGCAAGGCCAAC GTCATCGAAGTGATGGTCAAGGCCGGCGACACGGTCGAGGCCGACCAGTCGCTGATCACCCTGGAAT CCGACAAGGCCAGCATGGAGATCCCCTCGCCGGCCTCCGGGGTGGTGGAAAGCGTCTCGATCAAGGT CGGTGACGAAGTCGGCACCGGCGACCTGATCCTCAAGCTGAAGGTGGAAGGCGCCGCTCCGGCAGCC GAAGAGCAACCGGCAGCCGCTCCGGCCCAGGCCGCGGCGCCCGCCGCCGAGCAGAAGCCCGCCGCGG CGGCCCCTGCGCCAGCCAAGGCCGATACCCCGGCTCCGGTCGGCGCACCCAGCCGCGACGGCGCCAA GGTCCACGCCGGCCCGGCGGTGCGCATGCTGGCGCGCGAGTTCGGCGTCGAGCTGAGCGAAGTGAAA GCCAGCGGTCCCAAGGGTCGCATCCTCAAGGAAGACGTCCAGGTCTTCGTCAAGGAGCAACTGCAGC GCGCCAAGTCCGGCGGTGCCGGCGCCACCGGCGGAGCCGGCATCCCGCCGATCCCGGAAGTCGACTT CAGCAAGTTCGGCGAAGTGGAAGAAGTGGCGATGACCCGCCTGATGCAGGTCGGCGCCGCCAACCTG CATCGCAGCTGGCTGAACGTGCCGCACGTGACCCAGTTCGACCAGTCGGACATCACCGACATGGAAG CCTTCCGCGTTGCCCAGAAGGCCGCGGCGGAGAAGGCCGGGGTCAAGCTGACCGTACTGCCGATCCT GCTCAAGGCCTGCGCCCACCTGCTCAAGGAACTGCCGGACTTCAACAGTTCGCTGGCCCCCAGCGGC AAGGCGCTGATCCGCAAGAAGTACGTACACATCGGCTTCGCCGTGGACACTCCGGACGGCCTGCTGG TCCCGGTGATCCGCGATGTCGACCGGAAGAGCCTCCTGCAACTGGCCGCCGAGGCCGCCGACCTGGC CGACAAGGCCCGCAACAAGAAGCTCTCGGCCGATGCCATGCAGGGCGCCTGCTTCACCATCTCCAGT CTCGGCCACATCGGCGGCACCGGCTTCACGCCGATCGTCAACGCGCCGGAAGTGGCGATCCTCGGTG TGTCCAAGGCGACCATGCAGCCGGTATGGGACGGCAAGGCCTTCCAGCCGCGCCTGATGCTGCCGCT GTCGCTGTCCTACGACCATCGCGTGATCAACGGTGCCGCCGCGGCGCGCTTCACCAAGCGCCTGGGC GAGCTGCTGGCGGACATCCGCACCCTGCTCCTGTAA

The VIR7 protein (SEQ ID NO:14) encoded by SEQ ID NO:13 is presented using the one-letter amino acid code in Table 9B. TABLE 9B Encoded VIR7 protein sequence (SEQ ID NO: 14) MSELIRVPDIGNGEGEVIELLVKPGDKVEADQSLLTLESDKASMEIPSPKAGVVKSIKAKVGDTL KEGDEILELEVEGGEQPAEAKAEAAPAQPEAPKAEAPAPAPSESKPAAPAAASVQDIKVPDIGSA GKANVIEVMVKAGDTVEADQSLITLESDKASMEIPSPASGVVESVSIKVGDEVGTGDLILKLKVE GAAPAAEEQPAAAPAQAAAPAAEQKPAAAAPAPAKADTPAPVGAPSRDGAKVHAGPAVRMLAREF GVELSEVKASGPKGRILKEDVQVFVKEQLQRAKSGGAGATGGAGIPPIPEVDFSKFGEVEEVAMT RLMQVGAANLHRSWLNVPHVTQFDQSDITDMEAFRVAQKAAAEKAGVLKTVLPILLKACAHLLKE LPDFNSSLAPSGKALIRKKYVHIGFAVDTPDGLLVPVIRDVDRKSLLQLAAEAADLADKARNKKL SADAMQGACFTISSLGHIGGTGFTPIVNAPEVAILGVSKATMQPVWDGKAFQPRLMLPLSLSYDH RVINGAAAARFTKRLGELLADIRTLLL MUT8

A Pseudomonas bacterial mutant (MUT8) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding NADH dehydrogenase I chain H (nuoH; PA2643). This gene encodes the VIR8 nucleic acid (SEQ ID NO:15) shown in Table 10A. TABLE 10A VIR8 Nucleotide Sequence (SEQ ID NO: 15) ATGAGTTGGCTGACTCCCGCTCTGGTCACCATCATCCTCACCGTGGTCAAGGCCATCGTGGTGCTGC TCGCCGTGGTCATCTGCGGCGCCCTGCTAAGCTGGGTCGAGCGCCGCCTGCTCGGCCTCTGGCAGGA CCGCTACGGCCCCAACCGGGTCGGTCCGTTCGGTGCGTTCCAGCTCGGCGCGGACATGGTCAAGATG TTCTTCAAGGAGGACTGGACCCCGCCGTTCGCCGACAAGATGATCTTCACCCTGGCCCCGGTAATCG CGATGGGCGCCCTGCTCGTCGCCTTCGCCATCGTGCCGATCACCCCCACCTGGGGCGTGGCGGACCT GAACATCGGCATCCTGTTCTTCTTCGCCATGGCCGGCCTGACGGTGTACGCCGTGCTGTTCGCCGGC TGGTCGAGCAACAACAAGTTCGCCCTGCTCGGCAGCCTGCGCGCCTCGGCCCAGACCATCTCCTACG AGGTGTTCCTGGCCCTGTCGCTGATGGGCATCGTCGCCCAGGTCGGCTCGTTCAACATGCGCGACAT CGTCCAGTACCAGATCGACAACGTCTGGTTCATCATTCCGCAGTTCTTCGGCTTCTGCACCTTCATC ATCGCCGGCGTCGCCGTGACCCACCGTCACCCGTTCGACCAGCCGGAAGCGGAGCAGGAACTGGCGG ACGGCTACCACATCGAGTACGCCGGGATGAAATGGGGCATGTTCTTCGTCGGCGAGTACATCGGCAT CGTACTGGTCTCGGCGCTGCTGGCGACCCTGTTCTTCGGCGGCTGGCACGGTCCGTTCCTGGACACC CTGCCCTGGCTGTCGTTCTTCTACTTCGCCGCCAAGACCGGCTTCTTCATCATGCTCTTCATCCTGA TCCGCGCCTCGCTGCCGCGTCCGCGCTATGACCAGGTGATGGCGTTCAGCTGGAAGGTGTGCCTGCC GCTGACCCTGATCAACCTGCTGGTGACCGGCGCGCTCGTGCTGGCCGCGGCCCAGTAA

The VIR8 protein (SEQ ID NO:16) encoded by SEQ ID NO:15 is presented using the one-letter amino acid code in Table 10B. TABLE 10B Encoded VIR8 protein sequence (SEQ ID NO: 16) MSWLTPALVTIILTVVKAIVVLLAVVICGALLSWVERRLLGLWQDRYGPNRVGPFGAFQLGADMV KMFFKEDWTPPFADKMIFTLAPVIAMGALLVAFAIVPITPTWGVADLNIGILFFFAMAGLTVYAN LFAGWSSNNKFALLGSLRASAQTISYEVFLALSLMGIVAQVGSFNMRDIVQYQIDNVWFIIPQFF GFCTFIIAGVAVTHRHPFDQPEAEQELADGYHIEYAGMKWGMFFVGEYIGIVLVSALLATLFFGG WHGPFLDTLPWLSFFYFAAKTGFFIMLFILIRASLPRPRYDQVMAFSWKVCLPLTLINLLVTGAL VLAAAQ MUT9

A Pseudomonas bacterial mutant (MUT9) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding pyoverdine synthase D (PvdD; PA2399). This gene encodes the VIR9 nucleic acid (SEQ ID NO:17) shown in Table 11A. TABLE 11A VIR9 Nucleotide Sequence (SEQ ID NO: 17) GTGCAAGCACTCATAGAGAAGGTGGGCTCCCTTTCCCCCCAGGAAAGGAAGGCATTGGCTGTCCTGC TCAAGCAGCAAGGTGTCAATCTCTTCGAGATCGCGCCAGTGTTCAAGCGCCAGGACGGCGAGCCCCT GCGGCTCTCCTATGCCCAGGAGCGACAGTGGTTTCTCTGGCAACTGGAGCCGGAAAGCGCGGCCTAC CATATCCCGAGTGTCTTGCGTCTACGTGGGCGGCTGGACCTGGATGCCCTGCAACGCAGCTTCGACA GCCTGGTTGCGCGGCACGACACCCTACGCACCCGTTTTCGCCTCGACGGCGACGAGGCGCGCCAGGA GATCGCCGCATCCATGGCATTGCCGTTGGATATCGTCGCGTTGGGGCCGCTCGAGGAGGGCGCCCTC GCTCGGCAGGTCGAGACGACGATCGCGCGGCCGTTCGACCTGGAGCGTGGGCCGCTGCTGCGGGTGA GCCTGTTGCGGCTGGCCGAGGACGACCATGTGCTGGTGCTGGTCCAGCATCACATCGTGTCCGACGG TTGGTCGATGCAGGTGATGGTCGAGGAACTGGTCCAGCTCTATGCCGCCTATAGTCGAGGGCTCGAG GTAGCGCTGCCGGCTTTGCCGATCCAGTACGCGGACTACGCCCTGTGGCAGCGCAGCTGGATGGAGG CCGGGGAAAAGGAGCGCCAGTTGGCGTACTGGACCGGCCTGCTGGGCGGCGAGCAGCCGGTGCTGGA GTTGCCGTTCGACCGGCCCCGCCCGGTTCGGCAAAGCCATCGTGGTGCCCAGTTCATCCTGGAACTG GATATTGATCTGTCCCAGGCGCTCAGGCGCGTGGCCCAGCAGGAGGGGGCTACTGCCTTCGCCCTGT TGCTGGCTTCGTTCCAGGCGCTGCTGTATCGCTACAGCGGGCAGGCGGATATCCGTGTCGGCGTGCC GATCGCCAATCGCAACCGCGTGGAGACCGAGCGGCTGATCGGCTTCTTCGTCAACACCCAGGTGCTC AAGGCCGACCTGGACGGTCGGATGGGCTTCGACGAGCTGCTGGCCCAGGCCCGCCAACGCGCGCTGG AGGCCCAGGCGCACCAGGACCTGCCGTTCGAGCAACTGGTGGAGGCCTTGCAGCCGGAGCGCAGTCT TAGCCACAACCCGCTGTTCCAGGTGCTGTTCAACTACCAGAGCGAAGCCCGTGGCAACGGCCAGGCA TTCCGCTTCGACGAGTTACAGATGGAAAGCGTGCAGTTCGACAGCCGGACGGCGCAGTTCGACTTGA CGTTGGACCTGACGGACGAAGAGCAGCGTTTTTGCGCCGTTTTCGACTACGCCACCGACCTGTTCGA CGCCTCCACCGTGGAACGCCTGGCCGGCCATTGGCGCAACCTGTTGCGCGGCATCGTCGCCAACCCA CGACAGCGGCTCGGCGAGTTGCCGCTGCTGGATGCGCCGGAGCGCCGGCAGACCCTCTCCGAATGGA ACCCGGCCCAGCGCGAGTGCGCGGTGCAGGGCACCTTGCAGCAGCGTTTCGAGGAACAGGCGCGGCA ACGGCCACAGGCGGTTGCGCTGATCCTCGACGAACAACGGTTGAGCTACGGCGAACTGAATGCGCGG GCCAATCGCCTGGCGCACTGCCTGATCGCCCGTGGCGTTGGCGCGGACGTGCCGGTCGGGCTGGCGC TGGAGCGTTCGCTGGACATGCTGGTCGGCTTGCTGGCGATCCTCAAGGCCGGCGGCGCCTACCTGCC GTTGGACCCGGCGGCGCCAGAGGAGCGCCTGGCGCATATCCTCGACGACAGTGGGGTACGGCTGCTG CTGACCCAGGGGCATCTGCTCGAGCGCCTGCCACGGCAGGCGGGGGTGGAGGTGCTGGCCATCGACG GACTGGTGCTGGACGGCTACGCCGAGAGCGATCCGCTCCCGACGCTATCGGCGGACAACCTGGCCTA CGTGATCTATACCTCGGGCTCGACCGGCAAGCCCAAGGGCACATTGCTCACCCACCGCAACGCGCTG CGCCTGTTCAGCGCCACCGAGGCCTGGTTCGGCTTCGACGAGCGGGACGTGTGGACATTGTTCCATT CCTACGCCTTCGATTTCTCGGTCTGGGAAATCTTCGGCGCGCTGCTCTATGGCGGGTGCCTGGTGAT TGTGCCGCAATGGGTGAGCCGTTCGCCGGAAGACTTCTACCGTCTGCTGTGCCGCGAAGGCGTGACG GTGCTCAACCAGACGCCGTCGGCGTTCAAGCAACTGATGGCGGTGGCCTGTTCCGCCGACATGGCGA CGCAGCAGCCGGCGCTGCGCTACGTGATCTTCGGTGGCGAGGCGCTGGATCTGCAGAGCCTGCGGCC GTGGTTCCAGCGCTTCGGCGATCGCCAGCCGCAACTGGTGAACATGTACGGCATCACCGAGACCACG GTGCACGTAACCTACCGTCCGGTGAGCGAGGCCGACCTGGAAGGTGGCCTGGTCAGTCCGATTGGCG GGACCATCCCGGACCTGTCCTGGTACATCCTCGACCGTGACCTGAACCCGGTGCCGCGCGGCGCGGT GGGCGAGCTGTACATCGGTCGCGCCGGGCTGGCGCGCGGCTACCTGAGGCGGCCCGGGTTGAGTGCC ACCCGCTTCGTGCCGAACCCGTTCCCCGGCGGCGCCGGCGAGCGGCTGTACCGTACCGGCGACCTGG CACGGTTCCAGGCGGATGGCAATATCGAGTACATCGGGCGTATCGACCACCAGGTGAAGGTTCGCGG CTTCCGTATCGAACTGGGCGAGATCGAAGCGGCGCTCGCCGGTCTGGCCGGGGTACGCGATGCCGTG GTGCTGGCCCATGACGGAGTCGGCGGCACGCAACTGGTGGGATACGTGGTGGCGGACTCGGCGGAGG ATGCCGAGCGTCTGCGGGAGTCGCTGCGGGAGTCGCTGAAGCGGCACCTGCCGGACTACATGGTGCC GGCGCACCTGATGCTGCTGGAGCGGATGCCGCTGACGGTCAATGGCAAGCTCGACCGGCAGGCGTTG CCGCAACCGGATGCGAGCCTGTCGCAACAGGCCTATCGAGCGCCCGGTAGCGAGCTGGAGCAGCGCA TCGCAGCGATCTGGTCGGAGATCCTGGGAGTGGAACGGGTCGGCCTGGACGACAACTTCTTCGAACT GGGCGGTCATTCGTTGCTGGCTACCCGGGTGATTTCTCGGGTTCGCCAGGAGCAGCAGTTGGACGCA AGCCTGAAGGCGTTGTTCGAGCGGCCGGTTCTGGAAGCGTTCGCCCAGGGATTGGAACGCACGACGG ATGCGGTCTCGACGATACCGCTTGCCGATCGGCAGCAACCGTTGGCACTGTCCTTCGCTCAGGAGCG TCAGTGGTTCCTCTGGCAACTGGAGCCGGAAAGCGCGGCCTACCATATTCCGAGTGCCTTGCGCCTA CGCGGGCGGCTGGACGTGGATGCCTTGCAACGCAGCTTCGACAGCCTGGTCGCGCGGCATGAAACCT TGCGTACCCGCTTCCGGCTGGAGGGAGGGCGTTCGTACCAGCAGGTACAACCTGCGGTTAGCGTTTC CATCGAGCGGGAACAGTTCGGTGAAGAAGGCCTGATCGAACGGATACAGGCCATCGTTGTGCAGCCA TTCGACCTGGAACGGGGGCCGCTGCTGCGGGTGAACCTGTTGCAACTGGCCGAGGACGACCATGTAC TGGTGCTGGTCCAGCACCACATCGTGTCCGATGGTTGGTCGATGCAGGTGATGGTCGAGGAACTGGT CCAGCTCTATGCCGCCTATAGCCAAGGGCTCGACGTGGTGTTGCCAGCCCTGCCGATCCAGTACGCG GACTACGCCCTGTGGCAGCGCAGCTGGATGGAGGCGGGGGAAAAGGAGCGCCAGTTGGCGTACTGGA CCGGCCTGCTGGGCGGCGAGCAGCCGGTGCTGGAGTTGCCGTTCGATCGGCCGCGTCCGGCCCGGCA GAGCCATCGTGGCGCGCAGTTGGGTTTCGAGCTATCGCGGGAACTGGTCGAGGCCGTGAGAGCCTTG GCCCAGCGTGAAGGCGCCAGTAGTTTCATGCTGTTGCTGGCCTCGTTCCAGGCGCTGTTGTATCGCT ACAGCGGGCAGGCGGATATCCGTGTCGGTGTGCCGATCGCCAATCGCAACCGCGTGGAGACCGAGCG GCTGATCGGCTTCTTCGTCAACACCCAGGTGCTCAAGGCCGACCTGGACGGTCGGATGGGCTTCGAC GAGCTGCTGGCCCAGGCCCGCCAACGCGCGCTGGAGGCCCAGGCGCACCAGGACCTGCCGTTCGAGC AACTGGTGGAAGCCTTGCAGCCGGAGCGCAATGCCAGCCACAACCCACTGTTCCAGGTGCTGTTCAA CCATCAGAGCGAGATACGCTCGGTGACGCCCGAGGTTCAGTTGGAGGACCTGCGTCTGGAAGGCCTG GCCTGGGACGGCCAGACTGCGCAGTTCGACCTGACGCTGGATATTCAGGAAGACGAAAACGGCATCT GGGCCTCCTTCGACTATGCCACCGATCTGTTCGACGCCTCCACCGTGGAACGCCTGGCCGGCCATTG GCGCAACCTGTTGCGCGGCATCGTCGCCAACCCACGACAGCGGCTCGGCGAGTTGCCGCTGCTGGAT GCGCCGGAGCGCCGGCAGACCCTCTCCGAATGGAACCCGGCCCAGCGCGAGTGCGCGGTGCAGGGCA CCTTGCAGCAGCGTTTCGAGGAGCAGGCGCGGCAACGGCCACAGGCGGTTGCGCTGATCCTCGACGA ACAACGGTTGAGCTACGGCGAACTGAATGCGCGGGCCAATCGCCTGGCGCACTGCCTGATCGCTCGC GGCGTTGGCGCGGACGTGCCGGTCGGGCTGGCGCTGGAGCGTTCGCTGGACATGCTGGTCGGCTTGC TGGCGATCCTCAAGGCCGGCGGCGCCTACCTGCCGTTGGACCCGGCGGCGCCACAGGAGCGCCTGGC GCATATCCTCGACGACAGTGGGGTACGGCTGCTGCTGACCCAGGGGCATCTGCTCGAGCGCCTGCCG CGGCAGGCGGGGGTGGAGGTGCTGGCCATCGACGGACTGGTGCTGGACGGCTACGCCGAGAGCGATC CGCTCCCGACGCTATCGGCGGACAACCTGGCCTACGTGATCTATACCTCGGGCTCGACCGGCAAGCC CAAGGGCACGTTGCTCACCCACCGCAACGCGCTGCGCCTGTTCAGCGCCACCGAGGCCTGGTTCGGC TTCGACGAGCGGGACGTGTGGACGTTGTTCCATTCCTACGCCTTCGATTTCTCGGTCTGGGAAATCT TCGGCGCGCTGCTCTATGGCGGGCGCCTGGTGATCGTGCCGCAATGGGTGAGCCGTTCGCCGGAAGA CTTCTACCGTCTGCTGTGCCGCGAAGGCGTGACGGTGCTCAACCAGACGCCGTCGGCGTTCAAGCAA CTGATGGCGGTGGCCTGTTCCGCCGACATGGCGACGCAGCAGCCGGCGCTGCGCTACGTGATCTTCG GTGGCGAGGCGCTGGATCTGCAGAGCCTGCGGCCGTGGTTCCAGCGCTTTGGCGATCGCCAGCCGCA ACTGGTGAACATGTACGGCATCACCGAGACCACGGTACACGTAACCTACCGTCCGGTGAGCGAAGCC GACCTGAAGGGTGGCCTGGTCAGTCCGATCGGCGGGACCATCCCGGACCTGTCCTGGTACATCCTCG ACCGTGACCTGAACCCGGTGCCGCGCGGCGCGGTGGGCGAGCTGTACATCGGTCGCGCCGGTCTGGC GCGCGGCTACCTGAGGCGGCCCGGGTTGAGTGCCACCCGCTTCGTGCCGAACCCGTTCCCCGGCGGT GCCGGCGAGCGGCTGTACCGTACCGGCGACCTGGCACGGTTCCAGGCGGATGGCAATATCGAGTACA TCGGGCGTATCGACCACCAGGTGAAGGTTCGCGGCTTCCGTATCGAACTGGGTGAGATCGAAGCGGC GCTCGCCGGTCTGGCCGGGGTACGCGATGCCGTGGTGCTGGCCCATGACGGGGTCGGCGGCACGCAA CTGGTGGGATACGTGGTGGCGGACTCGGCGGAGGATGCCGAGCGTCTGCGGGAGTCGCTGCGGGAGT CGCTGAAGCGGCACCTGCCGGACTACATGGTGCCGGCGCACCTGATGCTGCTGGAGCGGATGCCGCT GACGGTCAATGGCAAGCTCGACCGGCAGGCGTTGCCGCAACCGGATGCGAGCTTGTCGCAGCAGGCC TATCGAGCGCCCGGTAGCGAGCTGGAGCAGCGCATCGCAGCGATCTGGGCGGAGATCCTGGGAGTGG AACGGGTCGGCCTGGACGACAACTTCTTCGAACTGGGCGGTCACTCATTGTTGCTGCTGATGCTCAA GGAGCGGATCGGCGATACCTGCCAGGCTACGCTGAGCATCAGCCAACTGATGACCCATGCCAGCGTC GCGGAACAGGCGGCATGCATCGAGGGGCAGGCGCGTGAGTCGTTGCTGGTGCCGCTCAACGGCAGGC GCGAAGGTTCGCCGCTGTTCATGTTCCATCCGAGTTTCGGCTCTGTGCACTGTTACAAGACCCTCGC CATGGCGCTGCGGGATCGTCATCCGGTCAAGGGTGTTGTCTGCCGTGCCCTGCTGGGCGCTGGTCGC GAGGTGCCGGAGTGGGACGATATGGTTGCGGAATACGCCGAGCAATTGCTGCAGGAGCACCCCGAAG GGGTTTTCAACCTGGCGGGATGGTCGCTCGGCGGCAACCTGGCGATGGATGTCGCGGCCCGGCTGGA GCAGCGTGGGCGGCAGGTGGCTTTCGTCGGCTGGATCGATGCACCGGCACCGGTCAGGGTCGAAGCG TTCTGGAACGAGATCGGGCCGACGCCGGAGGCAGTCCCGAACCTATCCGTGGGCGAGATGCGGGTGG AACTGCTCGGTGTCATGTTTCCGGAGCGGGCCGAGCATATCGAACGGGCCTGGTCATCGATCTGCTC CGCCACGACGGACGATGAGCAGCGCTGGACGAGGATGAGCGACTGGGCGGAAGCGGAGATCGGCGCC GAGTTCGCGACACTGCGCAGCGAAATCGCACAGAGCAACGAACTGGAAGTGTCCTGGGAGTTGAAAC AGATCCTCGACGAGCGCCTGAAAGCGATGGATTACCCGCGTCTGACGGCGAAGGTCAGCCTCTGGTG GGCCGCGCGCAGCACCAATGCCATCCAGCGGAGCGCGGTGGAGCGCTCGATGGCCGAGGCGATCGGG GCTGAGCGTGTCGAACCGGTGCGGGTGCTGGATACCCGGCACGACAAGATCATCGACCACCCTGAGT TTGTGCAGAGCTTCCGGGCCGCCCTGGAGCGTGCCGGGCGCTGA

The VIR9 protein (SEQ ID NO:18) encoded by SEQ ID NO:17 is presented using the one-letter amino acid code in Table 11B. TABLE 11B Encoded VIR9 protein sequence (SEQ ID NO: 18) MQALIEKVGSLSPQERKALAVLLKQQGVNLFEIAPVFKRQDGEPLRLSYAQERQWFLWQLEPESA AYHIPSVLRLRGRLDLDALQRSFDSLVARHETLRTRFRLDGDEARQEIAASMALPLDIVALGPLE EGALARQVETTIARPFDLERGPLLRVSLLRLAEDDHVLVLVQHHIVSDGWSMQVMVEELVQLYAA YSRGLEVALPALPIQYADYALWQRSWMEAGEKERQLAYWTGLLGGEQPVLELPFDRPRPVRQSHR GAQFILELDIDLSQALRRVAQQEGATAFALLLASFQALLYRYSGQADIRVGVPIANRNRVETERL IGFFVNTQVLKADLDGRMGFDELLAQARQRALEAQAHQDLPFEQLVEALQPERSLSHNPLFQVLF NYQSEARGNGQAFRFDELQMESVQFDSRTAQFDLTLDLTDEEQRFCAVFDYATDLFDASTVERLA GHWRNLLRGIVANPRQRLGELPLLDAPERRQTLSEWNPAQRECAVQGTLQQRFEEQARQRPQAVA LILDEQRLSYGELNARANRLAHCLIARGVGADVPVGLALERSLDMLVGLLAILKAGGAYLPLDPA APEERLAHILDDSGVRLLLTQGHLLERLPRQAGVEVLAIDGLVLDGYAESDPLPTLSADNLAYVI YTSGSTGKPKGTLLTHRNALRLFSATEAWFGFDERDVWTLFHSYAFDFSVWEIFGALLYGGCLVI VPQWVSRSPEDFYRLLCREGVTVLNQTPSAFKQLMAVACSADMATQQPALRYVIFGGEALDLQSL RPWFQRFGDRQPQVLNMYGITETTVHVTYRPVSEADLEGGLVSPIGGTIPDLSWYILDRDLNPVP RGAVGELYIGRAGLARGYLRRPGLSATRFVPNPFPGGAGERLYRTGDLARFQADGNIEYIGRIDH QVKVRGFRIELGEIEAALAGLAGVRDAVVLAHDGVGGTQLVGYVVADSAEDAERLRESLRESLKR HLPDYMVPAHLMLLERMPLTVNGKLDRQALPQPDASLSQQAYRAPGSELEQRIAAIWSEILGVER VGLDDNFFELGGHSLLATRVISRVRQEQQLDASLKALFERPVLEAFAQGLERTTDAVSTIPLADR QQPLALSFAQERQWFLWQLEPESAAYHIPSALRLRGRLDVDALQRSFDSLVARHETLRTRFRLEG GRSYQQVQPAVSVSIEREQFGEEGLIERIQAIVVQPFDLERGPLLRVNLLQLAEDDHVLVLVQHH IVSDGWSMQVMVEELVQLYAAYSQGLDVVLPALPIQYADYALWQRSWMEAGEKERQLAYWTGLLG GEQPVLELPFDRPRPARQSHRGAQLGFELSRELVEAVRALAQREGASSFMLLLASFQALLYRYSG QADIRVGVPIANRNRVETERLIGFFVNTQVLKADLDGRMGFDELLAQARQRALEAQAHQDLPFEQ LVEALQPERNASHNPLFQVLFNHQSEIRSVTPEVQLEDLRLEGLAWDGQTAQFDLTLDIQEDENG IWASFDYATDLFDASTVERLAGHWRNLLRGIVANPRQRLGELPLLDAPERRQTLSEWNPAQRECA VQGTLQQRFEEQARQRPQAVALILDEQRLSYGELNARANRLAHCLIARGVGADVPVGLALERSLD MLVGLLAILKAGGAYLPLDPAAPEERLAHILDDSGVRLLLTQGHLLERLPRQAGVEVLAIDGLVL DGYAESDPLPTLSADNLAYVIYTSGSTGKPKGTLLTHRNALRLFSATEAWFGFDERDVWTLFHSY AFDFSVWEIFGALLYGGRLVIVPQWVSRSPEDFYRLLCREGVTVLNQTPSAFKQLMAVACSADMA TQQPALRYVIFGGEALDLQSLRPWFQRFGDRQPQLVNMYGITETTVHVTYRPVSEADLKGGLVSP IGGTIPDLSWYILDRDLNPVPRGAVGELYIGRAGLARGYLRRPGLSATRFVPNPFPGGAGERLYR TGDLARFQADGNIEYIGRIDHQVKVRGFRIELGEIEAALAGLAGVRDAVVLAHDGVGGTQLVGYV VADSAEDAERLRESLRESLKRHLPDYMVPAHLMLLERMPLTVNGKLDRQALPQPDASLSQQAYRA PGSELEQRIAAIWAEILGVERVGLDDNFFELGGHSLLLLMLKERIGDTCQATLSISQLMTHASVA EQAACIEGQARESLLVPLNGRREGSPLFMFHPSFGSVHCYKTLAMALRDRHPVKGVVCRALLGAG REVPEWDDMVAEYAEQLLQEHPEGVFNLAGWSLGGNLAMDVAARLEQRGRQVAFVGWIDAPAPVR VEAFWNEIGPTPEAVPNLSVGEMRVELLGVMFPERAEHIERAWSSICSATTDDEQRWTRMSDWAE AEIGAEFATLRSEIAQSNELEVSWELKQILDERLKAMDYPRLTAKVSLWWAARSTNAIQRSAVER SMAEAIGAERVEPVRVLDTRHDKIIDHPEFVQSFRAALERAGR

A Pseudomonas bacterial mutant (MUT10) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding the RND multidrug efflux transporter MexD (mexD; PA4598). This gene encodes the VIR10 nucleic acid (SEQ ID NO:19) shown in Table 12A. TABLE 12A VIR10 Nucleotide Sequence (SEQ ID NO: 19) ATGTCCGAATTCTTCATCAAGCGGCCGAACTTCGCCTGGGTGGTGGCCCTGTTCATCTCCCTGGCCG GCCTGCTGGTCATTTCCAAATTGCCGGTAGCGCAGTACCCCAATGTCGCGCCGCCACAGATCACCAT CACCGCCACCTATCCCGGCGCCTCGGCGAAGGTGCTGGTGGACTCCGTCACCAGTGTGCTCGAGGAG TCGCTGAACGGCGCCAAGGGCCTGCTCTACTTCGAGTCGACCAACAACTCCAACGGCACCGCCGAGA TCGTCGTCACCTTCGAGCCGGGCACCGATCCGGACCTGGCCCAGGTGGACGTGCAGAACCGCCTGAA GAAAGCCGAGGCGCGCATGCCGCAGGCGGTGCTGACCCAGGGCCTGCAGGTCGAGCAGACCAGCGCC GGTTTCCTGCTGATCTATGCGCTCAGCTACAAGGAAGGCGCTCAGCGCAGCGACACCACCGCCCTCG GCGACTACGCCGCGCGCAATATCAACAACGAGCTGCGGCGCCTGCCGGGCGTCGGCAAGCTGCAATT CTTCTCTTCCGAGGCGGCCATGCGGGTCTGGATCGATCCGCAGAAGCTGGTGGGCTTCGGCCTCTCC ATCGACGACGTGAGCAATGCCATCCGCGGGCAGAACGTGCAGGTGCCGGCCGGCGCCTTCGGCAGCG CACCGGGCAGTTCCGCGCAGGAGCTGACGGCGACCCTGGCGGTGAAGGGCACCCTGGACGATCCGCA GGAGTTCGGCCAGGTAGTGCTGCGCGCCAACGAGGACGGCTCGCTGGTCCGGCTCGCCGATGTCGCG CGCCTGGAACTCGGCAAGGAGAGCTACAACATTTCCTCGCGACTGAACGGCACGCCCACCGTGGGCG GGGCTATCCAGCTGTCGCCCGGGGCCAACGCGATCCAGACCGCTACCCTGGTGAAACAGCGTCTCGC CGAACTGTCGGCGTTCTTCCCCGAGGACATGCAGTACAGCGTGCCCTACGACACCTCGCGCTTCGTC GACGTGGCCATCGAGAAGGTGATCCACACCCTGATCGAAGCGATGGTCCTGGTGTTCCTGGTGATGT TCCTGTTCCTGCAGAACGTCCGCTACACCCTGATCCCGTCCATCGTGGTGCCGGTGTGCCTGCTGGG TACGCTGATGGTGATGTACCTGCTGGGGTTCTCGGTGAACATGATGACCATGTTCGGCATGGTCCTG GCGATCGGCATCCTGGTGGACGACGCCATCGTGGTGGTGGAGAACGTCGAGCGGATCATGGCGGAGG AGGGGATTTCCCCGGCCGAGGCCACGGTCAAGGCGATGAAGCAGGTATCCGGCGCCATCGTCGGCAT CACCCTGGTGCTCTCGGCGGTGTTCCTGCCGCTGGCTTTCATGGCCGGTTCGGTGGGGGTGATCTAC CAGCAGTTCTCGGTGTCGCTGGCGGTCTCGATCCTGTTCTCCGGCTTCCTCGCCCTGACCTTCACCC CGGCGCTGTGCGCCACGCTGCTCAAGCCCATTCCCGAAGGGCACCACGAGAAGCGCGGCTTCTTCGG CGCCTTCAACCGTGGCTTCGCCCGCGTCACCGAGCGCTATTCGCTGCTCAACTCGAAGCTGGTGGCG CGCGCCGGACGCTTCATGCTGGTGTACGCCGGCCTGGTGGCCATGCTCGGCTACTTCTACCTGCGCC TGCCGGAAGCCTTCGTGCCGGCGGAAGACCTCGGCTACATGGTGGTCGACGTGCAACTGCCGCCTGG CGCTTCGCGCGTGCGCACCGATGCCACCGGCGAGGAGCTCGAGCGCTTCCTCAAGTCCCGCGAGGCG GTGGCTTCGGTGTTCCTGATCTCGGCTTCAGCTTCTCCGGCCAGGGCGACAATGCCGCCGCTGGCCT TCCCAACCTTCAAGGACTGGTCCGAGCGAGGCGCCGAGCAGTCGGCCGCCGCCGAGATCGCCGCGCT GAACGAGCATTTCGCGCTGCCCGACGATGGCACGGTCATGGCCGTGTCGCCGCCACCGATCAACGGT CTGGGTAACTCCGGCGGCTTCGCATTGCGCCTGATGGACCGTAGCGGGGTCGGCCGCGAAGCGCTGC TGCAGGCTCGCGATACTCTTCTTGGCGAGATCCAGACCAACCCGAAATTCCTTTACGCGATGATGGA AGGACTGGCCGAAGCGCCGCAACTGCGCCTGTTGATCGACCGGGAGAAGGCCCGTGCCCTGGGGGTG AGCTTCGAGACCATCAGCGGCACGCTGTCCGCTGCCTTCGGCTCGGAGGTGATCAACGACTTCACCA ATGCGGGGCGCCAACAGCGGGTGGTGATCCAGGCCGAACAGGGCAACCGGATGACCCCGGAAAGCGT GCTCGAGCTATACGTGCCTAACGCTGCTGGCAACCTGGTACCGCTCAGCGCCTTCGTCAGCGTGAAA TGGGAAGAGGGACCGGTGCAATTGGTGCGCTATAACGGCTACCCGTCGATCCGCATCGTCGGTGACG CCGCGCCCGGCTTCAGTACCGGCGAAGCCATGGCGGAAATGGAGCGCCTGGCCTCGCAGCTGCCGGC CGGCATCGGCTACGAGTGGACCGGCCTGCTCCTATCAGGAGAAGGTCTCCGCCGGCAGGCCACCAGC CTGTTCGCCCTCGCCATCCTGGTGGTGTTCCTGTTGCTGGTGGCGCTCTACGAGAGCTGGTCGATCC CGCTGTCGGTGATGCTGATCGTGCCGATCGGCGCCATCGGCGCGGTGCTCGCGGTGATGGTCAGCGG TATGTCCAACGACGTGTATTTCAAGGTCGGCCTGATCACCATCATCGGTCTTTCGGCGAAGAACGCG ATCCTCATCGTCGAGTTCGCCAAGGAACTCTGGGAGCAGGGGCATAGCCTGCGCGACGCCGCCATCG AGGCCGCGCGCCTGCGCTTCCGGCCGATCATCATGACTTCCATGGCGTTCATCCTCGGCGTGATACC CCTGGCCCTGGCCAGCGGTGCCGGCGCGGCGAGCCAGCGTGCCATCGGCACCGGAGTGATCGGCGGG ATGCTCAGCGCCACCTTCCTCGGCGTGCTGTTCGTACCTATCTGTTTCGTCTGGCTGCTGTCGCTGC TGCGCAGCAAGCCGGCACCCATCGAACAGGCCGCTTCGGCCGGGGAGTGA

The VIR10 protein (SEQ ID NO:20) encoded by SEQ ID NO:19 is presented using the one-letter amino acid code in Table 12B. TABLE 12B Encoded VIR10 protein sequence (SEQ ID NO: 20) MSEFFIKRPNFAWVVALFISLAGLLVISKLPVAQYPNVAPPQITITATYPGASAKVLVDSVTSVL EESLNGAKGLLYFESTNNSNGTAEIVVTFEPGTDPDLAQVDVQNRLKKAEARMPQAVLTQGLQVE QTSAGFLLIYALSYKEGAQRSDTTALGDYAARNINNELRRLPGVGKLQFFSSEAAMRVWIDPQKL VGFGLSIDDVSNAIRGQNVQVPAGAFGSAPGSSAQELTATLAVKGTLDDPQEFGQVVLRANEDGS LVRLADVARLELGKESYNISSRLNGTPTVGGAIQLSPGANAIQTATLVKQRLAELSAFFPEDMQY SVPYDTSRFVDVAIEKVIHTLIEAMVLVFLVMFLFLQNVRYTLIPSIVVPVCLLGTLMVMYLLGF SVNMMTMFGMVLAIGILVDDAIVVVENVERIMAEEGISPAEATVKAMKQVSGAIVGITLVLSAVF LPLAFMAGSVGVIYQQFSVSLAVSILFSGFLALTFTPALCATLLKPIPEGHHEKRGFFGAFNRGF ARVTERYSLLNSKLVARAGRFMLVYAGLVAMLGYFYLRLPEAFVPAEDLGYMVVDVQLPPGASRV RTDATGEELERFLKSREAVASVFLISGFSFSGQGDNAALAFPTFKDWSERGAEQSAAAEIAALNE HFALPDDGTVMAVSPPPINGLGNSGGFALRLMDRSGVGREALLQARDTLLGEIQTNPKFLYAMME GLAEAPQLRLLIDREKARALGVSFETISGTLSAAFGSEVINDFTNAGRQQRVVIQAEQGNRMTPE SVLELYVPNAAGNLVPLSAFVSVKWEEGPVQLVRYNGYPSIRIVGDAAPGFSTGEAMEAMERLAS QLPAGIGYEWTGLSYQEKVSAGQATSLFALAILVVFLLLVALYESWSIPLSVMLIVPIGAIGAVL AVMVSGMSNDVYFKVGLITIIGLSAKNAILIVEFAKELWEQGHSLRDAAIEAARLRFRPIIMTSM AFILGVIPLALASGAGAASQRAIGTGVIGGMLSATFLGVLFVPICFVWLLSLLRSKPAPIEQAAS AGE MUT11

A Pseudomonas bacterial mutant (MUT11) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding PA3721. This gene encodes the VIR11 nucleic acid (SEQ ID NO:21) shown in Table 13A. TABLE 13A VIR11 Nucleotide Sequence (SEQ ID NO: 21) ATGAACGATGCTTCTCCCCGTCTGACCGAACGCGGCAGGCAACGCCGCCGCGCCATGCTCGACGCCG CTACCCAGGCCTTTCTCGAACACGGTTTCGAAGGCACCACCCTGGACATGGTGATAGAACGGGCCGG TGGTTCACGGGGGACCCTGTACAGCTCCTTCGGCGGCAAGGAGGGCCTGTTCGCCGCGGTGATCGCC CACATGATCGGGGAAATCTTCGACGACAGCGCCGATCAGCCGCGCCCCGCCGCCACGCTGAGCGCCA CCCTCGAGCATTTCGGCCGGCGCTTTCTCACCAGCCTGCTCGATCCCCGCTGCCAGAGCCTCTATCG CCTGGTGGTGGCGGAATCCCCGCGGTTTCCGGCGATCGGCAAGTCCTTCTACGAGCAGGGGCCGCAG CAGAGCTATCTGCTGCTCAGCGAGCGACTGGCCGCGGTCGCTCCTCACATGGACGAGGAAACGCTCT ACGCGGTGGCCTGCCAGTTTCTCGAGATGCTCAAGGCCGACCTGTTCCTCAAGGCCCTCAGCGTGGC CGACTTCCAGCCGACCATGGCGCTGCTGGAAACCCGCCTCAAGCTGTCGGTGGACATCATCGCCTGC TACCTGGAACACCTGTCGCAGAGCCCCGCGCAGGGCTGA

The VIR11 protein (SEQ ID NO:22) encoded by SEQ ID NO:21 is presented using the one-letter amino acid code in Table 13B. TABLE 13B Encoded VIR11 protein sequence (SEQ ID NO: 22) MNDASPRLTERGRQRRRAMLDAATQAFLEHGFEGTTLDMVIERAGGSRGTLYSSFGGKEGLFAAV IAHMIGEIFDDSADQPRPAATLSATLEHFGRRFLTSLLDPRCQSLYRLVVAESPRFPAIGKSFYE QGPQQSYLLLSERLAAVAPHMDEETLYAVACQFLEMLKADLFLKALSVADFQPTMALLETRLKLS VDIIACYLEHLSQSPAQG MUT12

A Pseudomonas bacterial mutant (MUT12) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding PA0596. This gene encodes the VIR12 nucleic acid (SEQ ID NO:23) shown in Table 14A. TABLE 14A VIR12 Nucleotide Sequence (SEQ ID NO: 23) ATGTCTGATGATGCCCGTTTCCAGCAGCTGAATTGCTGGTTGGACTCTTGTTTGCCCGAGTTGTTCG TTGCCGAAGGTTGGGGGGAAGTGCCCCCCGCCGAACTGATCCCGGCCAGTAGCGACGCCAGCTTCCG TCGTTATTTCCGCTGGCAGGGAGGGGACCGCAGCCTGGTGGTGATGGACGCGCCGCCGCCCCAGGAA GACTGCCGACCGTTCGTCAAGGTCGCCGGACTGCTCGCCGGAGCCGGCGTGCATGTGCCGAGGATTC TCGCCCAGGACCTGGAGAACGGTTTCCTGCTGCTCAGTGACCTGGGCCGGCAGACCTACCTCGACGT GCTTCATCCCGGGAATGCCGACGAGCTGTTCGAACCGGCCCTGGATGCGCTGATCGCCTTCCAGAAG GTCGATGTCGCCGGTGTCCTGCCTGCCTACGACGAAGCGGTGCTGCGCCGCGAGCTGCAGCTGTTCC CCGACTGGTACCTGGCCCGCCACCTCGGCGTGGAGCTGGAGGGCGAGACGCTGGCCCGCTGGAAACG GATCTGCGACCTGCTGGTACGCAGCGCGCTGGAGCAACCGCGGGTGTTCGTCCATCGCGACTATATG CCGCGCAATCTGATGCTCAGCGAGCCCAACCCGGGCGTCCTCGACTTCCAGGACGCCCTGCACGGCC CGGTCACCTACGATGTCACCTGCCTGTACAAGGACGCCTTCGTCAGTTGGCCGGAGCCGCGCGTGCA TGCCGCGCTGAACCGTTACTGGAAGAAGGCGACCTGGGCCGGCATCCCGCTGCCGCCAAGCTTCGAA GACTTCCTCCGTGCCAGCGACCTGATGGGCGTGCAGCGCCACCTGAAGGTGATTGGCATCTTCGCCC GTATCTGTCACCGCGACGGCAAGCCGCGCTACCTGGGTGACGTGCCGCGCTTCTTCCGTTATCTGGA AACCGCCGTGGCGCGCCGTCCCGAGCTGGCCGAACTGGGCGAGCTGCTGGCCTCGCTGCCGCAGGGA GCCGAGGCATGA

The VIR12 protein (SEQ ID NO:24) encoded by SEQ ID NO:23 is presented using the one-letter amino acid code in Table 14B. TABLE 14B Encoded VIR12 protein sequence (SEQ ID NO: 24) MSDDARFQQLNCWLDSCLPELFVAEGWGEVPPAELIPASSDASFRRYFRWQGGDRSLVVMDAPPP QEDCRPFVKVAGLLAGAGVHVPRILAQDLENGFLLLSDLGRQTYLDVLHPGNADELFEPALDALI AFQKVDVAGVLPAYDEAVLRRELQLFPDWYLARHLGVELEGETLARWKRICDLLVRSALEQPRVF VHRDYMPRNLMLSEPNPGVLDFQDALHGPVTYDVTCLYKDAFVSWPEPRVHAALNRYWKKATWAG IPLPPSFEDFLRASDLMGVQRHLKVIGIFARICHRDGKPRYLGDVPRFFRYLETAVARRPELAEL GELLASLPQGAEA MUT13

A Pseudomonas bacterial mutant (MUT13) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding PA5265. This gene encodes the VIR13 nucleic acid (SEQ ID NO:25) shown in Table 15A. TABLE 15A VIR13 Nucleotide Sequence (SEQ ID NO: 25) ATGAGCGGATTCCAGGACCAGAGTATCGACGAAGGCGTGCGCAAGCGCACCGCCTACCAGAACGATC GGCGTGCACGACTGGCATTGAACGTCGAGCGACAGGACGGCGGTATCCTGCAGATTCCGGTGGCCAG CGATATGCTCGGCCATGAGGAGCACGAGCGTATCCAGCAGAACACCTTCCTGGCTGTGATGCCGCTG GTCCGCCTGCCAACGCTGGGCAAGGCCGGTTATGGCGACCAGCTGCCCGCCGGCGCGCTACCGCGGG CGGGACGGATCTACCTGTTCCAGGACGGCAAGTTGTGGCGCGAACTGGAATGTGATGGCAAGGGCAA CCTGTTCGAAGTCGATCTCCTGCAGGGGCGCAGCCAGCGTGCGGACAAGCGTCCGGCCTTAGGCAAG ACACAAGCGCTGATCCTGGTGCCGGTGCTGGTCAAGGGGCAGTTCGTGATCCCACGCTACACCATGG CCTATAGCGAAACTCCCTGGCCTTGGTCGTACATCGACTGGCTGGAGGAGGACCCGCAGCGGGTCAA CCGGCGCTGCCAGCAGATGGCGTCCGCTTGGAACGCCTCGGTGGCCAACCAGCACTGGAAAGCCTCC ATCCATCAACCCGCGCTGGTCATTGATCATCACGCCCAGGGTTTGCGACCTCGCGACTTCAACGTCG AGAGCGCGCTGGAAGACCCGGCGGAATTCACACCTGAGTTCGCCGCCTTTCGCGAAGAGTCGCTGGT GTGCCAGTTGCAGCGACGCCAGCAGGAATTGGCGCCCCTGCTGAAGCAGGCTCCGCCCTCTGCGCTA CCTACTCTGGAAGCCGGAGAGGACGTACTGGAAACCCTCAAGCTGCGTGGCCATCCCAACCTCATCG GGCTGATGCTCGACGACTCGCTGTTCGCCTTGCGCCACGCTGCGGCGCAGGCGCGCCACTGCGCCGC CTACTTGCGCAGCCTCAATGCACTGCTGCCGCACCGTCCCAACGGACGCTATGCACAGGTGCTGAGC AACATGCTCGACGGCCCGCTCGCCAAGCTCAGGGGCGAGGTCGATCAGGCCGAACTGGACGAGGCGA TCTTCGCCGAGGAGCGACAGTCTTGCCGAATCCACCTGACGCAGCAGGTCGAGCATCTGGTTGCCCT GCTGGAAGGCCCCTTGCACCCGGTGTTGCAGGACTGGACCCACCAGTGCGACGAAGCCCTGCTGGAG CCCTACAGCCTGATGAGCGAGGCACTGGCTGCGCTGAACCAGCTTCCCGACCGCTGCGACGCACTGT ACAGCGGTACCGCCTACCGGGCGCTGGCGGCACATGTCGAGCGGGTGGTCAGCACGGTTCTGCAGGC AAGCCACCCGCTTGGCGCCATGCTCCTGGCCAAGGACGAAGGACAACTTCCCGAGCCGGTTCGGCGC CTGCAGGCGCTGCGCGATAGCCCGCGGACGCCGGACCCCGATGCAATGGGCCTCAGCACGCTGATGC TGGGAGCCAGTCTGCTGGGCGAGGTCGACCAGCCCAGCGCCGGCAAGAGCCTCGCCTACTTCCTCGG CGACCTGCTGGACGTGTTCGGCGCCAGCGTAGTCGAGCAACTCGGCCGGCTGTCCCAGGGCGCCACC CAGATCCAGCTCGACCGCTTGTTCGCACCGACCTTCAATACTCTGAGCGCCCTCTCGGTGAAGATGA AAGGTATCCGCCTGCTGCCCGACAGTCAGGTGCCGCTCGACATGGTTGTCGTCGGCGTGCGCGGAGC CGGCCTGCGCAACGGTCTGACCGAGGTCGAGCGCCAGGAGCTGAGGCGCAAGAGCTATCGGCGCGCC ATCGTTCAGGACGGTGCCGGCAATCCCCTGGCCGGCACCAGTCCCCGCGACACCGGCATGAGTCGCG CCAACCTGCGCAACGTCATGGTGGTGGCGGTACCCAAGGATCACCCGGACCTGCTTGCCTACACGAA ATTCCGTACGCAGTTAGGCACGTTGACCCAGGTGATGGAGAACACTCGCATCGTGCCGACGATGATG CTGGGGTTTGCGATTTATAACTTGAATGTGCAGGTGCAGGCATACAGTGGCTTTGTAGACAGTGGAG AAAAGCACAGAGGGACGATCGGGGCTGTCGGTGCAGTAATCGATTTAACAGCCGCTGGAGGAAGCCA TGCAAAGCTGCTTTTCGGACCATCTACTGCAAAGTATCTAGAAACCCCACGTATATCGGTAGCCCAA ATATCCCCTCGATGGGCCAGGAATCTAGAAGTTCAAACAGGCAGCCCTAAGTTAGGGTTGCTACGTG GGCTTGGTGGCGCAGCCACACTATTCGGTGCAGGCATCAGTGTATGGGATGGCTACCGAGCTTTGAG GCAGGGAGATAGCGATGCGGCTGCGGCCTACGGTGTGGCCGCAGTGGGTGGGGGCCTTTGGGGTGCC TACGTCCTAGGATGGATAGTAAACCCTTATGCTTTGCTGGCTGGTGCGGTTTTGGCGATCGGAGGCA CTGTGGTCGCTAATCTACTGACTGACAGCGATGCGGAAACCATCGTAAAGAAAGGCCCCTTCGGCCG GCAATTCGCCGAGGCTGGCCTGCTCGATTCGCTGATGGGCCAGGACCAGCGCTTCGCCCATCTGAAA GACCCGCAAACGGCCTATCGCCAATTGCTGGGAGTCCTCGGCCATCCGCGGGTCTTTGTCCATCGCC TGGAAGACTGGCGCAAATTGGCGCCGGCGGCGCATCGATCTGTCTTGCAGGAAGCGGAACGGGGTCG CCAAGCGGTCAGCCGCACTGCGCTATCCTGCATCGACCCCAAGTTGCAGGCGCTGGAGGCAAACGAT TGGGCCGTGGTGCTGAGTTCCCCGCTCCTGGCCATGTTCGAGAATGGCCAGAAGGCGTTCCGCCTGG TGGCCCAGGAGTTTCTCAGCAGCTTGCCGATCGATCCGGGCACCCTGTTCGGCGTCAAGCGCTACCA TCGGGTCCCCGCGGGCCCCGCCAAGCTCGAAGCCTTGCCGTTGGATGCTGCCAGCGTGCTCTATGTG CTGCCGGCCAGCCTGCCGATTCCGCAGTTGTCTCCTCGGGCCCGCTATAGCATGCGCATGACCCAGG GTTTGAAGATCAGCGCACAGTTCGAACTCAATGCCGACCAGCCTGAGCAGCGGCTTGTCCTGCCTCA ACCCAGCCCGAAGAGTTGGAGTGCATTCACATCCGCCAATCGGTACCTTCCCCCGGACGACTTGGGC CCCCATGCTGCGCCACCTTATTGGTTGATAGAGAACAGTGAGTTCAACGTATGA

The VIR13 protein (SEQ ID NO:26) encoded by SEQ ID NO:25 is presented using the one-letter amino acid code in Table 15B. TABLE 15B Encoded VIR13 protein sequence (SEQ ID NO: 26) MSGFQDQSIDEGVRKRTAYQNDRRARLALNVERQDGGILQIPVASDMLGHEEHERIQQNTFLAVM PLVRLPTLGKAGYGDQLPAGALPRAGRIYLFQDGLKWRELECDGKGNLFEVDLLQGRSQRADKRP ALGKTQALILVPVLVKGQFVIPRYTMAYSETPWPWSYIDWLEEDPQRVNRRCQQMASAWNASVAN QHWKASIHQPALVIDHHAQGLRPRDFNVESALEDPAEFTPEFAAFREESLVCQLQRRQQELAPLL KQAPPSALPTLEAGEDVLETLKLRGHPNLIGLMLDDSLPALRHAAAQARHCAAYLRSLNALLPHR PNGRYAQVLSNMLDGPLAKLRGEVDQAELDEAIFAEERQSCRIHLTQQVEHLVALLEGPLHPVLQ DWTHQCDEALLEPYSLMSEALAALNQLPDRCDALYSGTAYRALAAHVERVVSTVLQASHPLGAML LAKDEGQLPEPVRRLQALRDSPRTPDPDAMGLSTLMLGASLLGEVDQPSAGKSLAYFLGDLLDVF GASVVEQLGRLSQGATQIQLDRLFAPTFNTLSALSVKMKGIRLLPDSQVPLDMVVVGVRGAGLRN GLTEVERQELRRKSYRRAIVQDGAGNPLAGTSPRDTGMSRANLRNVMVVAVPKDHPDLLAYTKFR TQLGTLTQVMENTRIVPTMMLGFAIYNLNVQVQAYSGFVDSGEKHRGTIGAVGAVIDLTAAGGSH AKLLFGPSTAKYLETPRISVAQISPRWARNLEVQTGSPKLGGLRGLGGAATLFGAGISVWDGYRA LRQGDSDAAAAYGVAAVGGGLWGAYVLGWIVNPYALLAGAVLAIGGTVVANLLTDSDAETIVKKG PFGRQFAEAGLLDSLMGQDQRFAHLKDPQTAYRQLLGVLGHPRVFVHRLEDWRKLAPAAHRSVLQ EAERGRQAVSRTALSCIDPKLQALEANDWAVVLSSPLLAMFENGQKAFRLVAQEFLSSLPIDPGT LFGVKRYHRVPAGPAKLEALPLDAASVLYVLPASLPIPQLSPRARYSMRMTQGLKISAQFELNAD QPEQRLVLPQPSPKSWSAFTSANRYLPPDDLGPHAAPPYWLIENSEFNV MUT14

A Pseudomonas bacterial mutant (MUT14) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding pyochelin biosynthetic protein pchC (PA4229). This gene encodes the VIR14 nucleic acid (SEQ ID NO:27) shown in Table 16A. TABLE 16A VIR14 Nucleotide Sequence (SEQ ID NO: 27) ATGAGCGCCGCCTGGGTCCGGCCGTTCCGCCTGACGCCGATGCCGCGCCTGCGCCTGGCCTGCTTCC CCCATGCAGGCGGCAGCGCCAGCTTCTTCCGTAGCTGGAGCGAACGCCTGCCGCCAGACATCGACCT GCTTGCCCTGCAGTACCCGGGTCGCGAGGACCGCTTCAACGAGGCGCCGGCCACCCGCCTGGAGGAC CTCGCCGACGGCGCCGCCCTCGCCCTGCGCGATTTCGCCGACGCGCCCCTGGCGCTGTTCGGCCACA GTCTCGGCGCGGCGCTGGCCTACGAAACCGCCCTGCGCCTGGAAAGCGCCGGCGCGCCGCTGCGCCA CCTGTTCGTCTCCGCCCATCCGGCACCGCACCGGCAACGCGGCGGCGCGTTGCACCGCGGCGACGAG GCGGCGCTGCTGGAGGACGTCCGCCGCCAGGGTGGCGCCAGCGAGCTACTCGAGGACGCCGACCTGC GCGCGCTGTTCCTGCCGATCCTGCGCGCCGACTACCAGGCGATCGAGACCTACCGACGGGCGCAGCC CATCGCCCTGGCCTGCGCCCTCGACGTCCTCCTCGGCGAGCACGACGAGGAAGTCAGCGCCGCCGAG GCGCAGGCCTGGAGCGACGCCAGCCGGACTCCCGCCAGGCTGCGGCGCTTTCCTGGCGGCCACTTCT ACCTGAGCGAGGGGCGCGACGCGGTGATCGAGCACCTGCTGCGCCGCCTCGCACATCCCGACGCCCT TTCCCGAGAGGTTGCATGA

The VIR14 protein (SEQ ID NO:28) encoded by SEQ ID NO:27 is presented using the one-letter amino acid code in Table 16B. TABLE 16B Encoded VIR14 protein sequence (SEQ ID NO: 28) MSAAWVRPFRLTPMPRLRLACFPHAGGSASFFRSWSERLPPDIDLLALQYPGREDRFNEAPATRLEDL ADGAALALRDFADAPLALFGHSLGAALAYETALRLESAGAPLRHLFVSAHPAPHRQRGGALHRGDEAA LLEDVRRQGGASELLEDADLRALFLPILRADYQAIETYRRAQPIALACALDVLLGEHDEEVSAAEAQA WSDASRTPARLRRFPGGHFYLSEGRDAVIEHLLRRLAHPDALSREVA MUT15

A Pseudomonas bacterial mutant (MUT15) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding dihydroaeruginoic acid synthetase pchE (PA4226). This gene encodes the VIR15 nucleic acid (SEQ ID NO:29) shown in Table 17A. TABLE 17A VIR15 Nucleotide Sequence (SEQ ID NO: 29) ATGGATCTGCCCCCCGATTCCCGTACCGCCCTGCGCGACTGGCTGACCGAGCAGCTCGCCGACCTGC TCGGCGAACCGCTTGCTGACGTGCGCGCCCTGGCGGACGACGACGACCTGCTGGGCTGCGGCCTCGA CTCGATCCGCCTGATGTACCTGCAGGAACGCCTGCGCGCGCGTGGCTCGACGCTGGACTTCGCCCAG TTGGCGCAGCGCCCCTGCCTGGGGGCCTGGCTCGACCTGCTGGCCTGCGCGGACCGGCTGTCCGCCC CGGCAACGGTCGCGCTGCCGACGGCGCAGGATCGCGATCAGCCGTTCGAGCTGTCTTCCGTGCAGCA GGCCTACTGGCTGCGACGTGGCGCCGGCGAGGTGCTGGGCAACGTCAGCTGCCATGCCTTTCTGGAA TTCCGCACGCGGGATGTCGACCCGCAGCGCCTGGCCGCGGCGGCGGAGTGCGTGCGTCAACGCCACC CGATGTTGCGGGCGCGCTTCCTCGACGGTCGCCAGCAGATCCTTCCGACGCCGCCGCTGTCCTGCTT CGACCTGCAGGACTGGCGCACCTTACAGGTGGACGAGGCCGAGCGCGACTGGCAGGCGCTGCGCGAC TGGCGCGCCCATGAATGCCTGGCGGTGGAGCGCGGCCAGGTGTTCCTGCTCGGGCTGGTGCGCATGC CGGGCGGCGAGGATCGCCTCTGGCTGAGTCTCGACCTGCTTGCCGCCGATGTCGAAAGCCTCCGCCT GCTGCTGGCCGAACTGGGCGTTGCCTACCTGGCGCCGGAGCGCCTGGCGGAGCCGCCGGCGCTGCAT TTCGCCGACTACCTGGCGCACCGTGCGGCGCAACGCGCCGAGGCCGCGGCGCGGGCCCGCGACTACT GGCTGGAACGCCTGCCGCGCTTGCCGGACGCGCCGGCCCTGCCGTTGGCCTGCGCGCCGGAAAGCAT CCGCCAGCCGCGCACCCGGCGCCTGGCATTCCAGCTTTCCGCCGGCGAGAGCCGGCGCCTGGAGCGT CTTGCCGCGCAGCATGGCGTGACCTTGTCCAGCGTGTTCGGCTGCGCCTTCGCGCTGGTCCTGGCGC GCTGGAGCGAAAGCGCGGAATTTCTCCTCAACGTGCCGTTGTTCGATCGGCATGCCGACGACCCGCG TATCGGCGAGGTGATCGCCGACTTCACCACCCTGTTGCTGCTGGAGTGCCGGATGCAGGCCGGGGTG TCCTTCGCCGAGGCGGTGAAGAGCTTCCAGCGCAACCTCCACGGAGCCATCGACCACGCCGCATTCC CCGCCCTGGAGGTGCTCCGCGAGGCGCGCCGGCAGGGCCAGCCACGCTCGGCGCCGGTGGTGTTCGC CAGCAACCTGGGCGAGGAGGGCTTCGTCCCGGCGGCCTTCCGCGACGCTTTCGGCGATCTCCACGAC ATGCTCTCGCAGACCCCGCAGGTCTGGCTCGACCACCAGCTCTACCGGGTGGGCGACGGTATCCTGC TGGCCTGGGATAGCGTCGTCGGCCTGTTCCCCGAAGGTCTGCCGGAAACCATGTTCGAAGCCTACGT GGGGCTGCTCCAGCGTCTCTGCGACAGCGCCTGGGGGCAGCCCGCCGATCTGCCGTTGCCCTGGGCG CAGCAGGCGCGCCGGGCCCTGCTCAACGGCCAGCCGGCATGCGCCACGGCGCGCACCCTGCATCGCG ACTTCTTCCTTCGCGCCGCCGAGGCGCCGGATGCCGACGCGCTGCTCTATCGCGACCAACGTGTCAC CCGCGGCGAACTGGCCGAGCGTGCGCTGCGCATCGCCGGCGGCCTGCGCGAAGCCGGGGTGCGCCCT GGCGACGCGGTCGAGGTCAGCCTGCCGCGCGGACCGCAGCAGGTCGCGGCGGTATTCGGCGTGCTCG CCGCAGGCGCCTGCTACGTGCCGCTGGACATCGACCAGCCGCCCGCACGGCGGCGCCTGATCGAAGA GGCCGCCGGGGTATGCCTGGCGATCACCGAGGAGGACGATCCGCAGGCCTTGCCGCCGCGCCTGGAT GTCCAGCGCCTGCTGCGCGGCCCGGCGCTGGCCGCCCCCGTGCCGCTGGCGCCGCAGGCGAGTGCCT ATGTGATCTACACCTCGGGCTCCACCGGGGTGCCCAAGGGCGTCGAGGTCAGCCACGCGGCGGCGAT CAATACCATCGACGCGCTGCTCGACCTGCTGCGGGTGAACGCATCGGATCGCTTGCTGGCGGTCTCC GCGCTGGACTTCGATCTGTCGGTCTTCGACCTGTTCGGCGGCCTCGGCGCCGGTGCCAGCCTGGTCC TGCCGGCCCAGGAACAGGCGCGCGATGCCGCTGCCTGGGCGGAGGCTATCCAGCGGCATGCGGTGAG CCTGTGGAACTCGGCGCCGGCCTTGCTGGAGATGGCCCTCAGCCTGCCGGCGAGCCAGGCCGACTAT CGCAGTCTGCGGGCGGTGCTGCTGTCCGGCGACTGGGTGGCCCTGGACCTGCCCGGCCGCCTGCGCC CACGTTGTGCCGAAGGCTGCCGCCTGCATGTGCTGGGTGGCGCTACCGAAGCGGGCATCTGGTCGAA CCTGCAGAGCGTCGATACGGTGCCGCCGCACTGGCGTTCGATTCCCTACGGCCGGCCATTGCCGGGA CAGGCCTACCGGGTGGTCGACACCCACGGGCGCGACGTGCCGGACCTGGTGGTCGGCGAGCTGTGGA TCGGCGGCGCCAGCCTGGCCCGCGGCTATCGCAACGATCCCGAACTCAGCGCCCGGCGTTTCGTCCA CGATGCCCAGGGCCGCTGGTATCGCACCGGCGATCGCGGTCGCTACTGGGGCGACGGTACCCTGGAA TTCCTCGGTCGGGTCGACCAGCAGGTGAAAGTGCGCGGCCAGCGCATCGAGTTGGGCGAGGTGGAGG CCGCGCTGTGCGCCCAGGCTGGCGTGGAGAGCGCCTGCGCGGCGGTGCTCGGCGGTGGCGTGGCGAG CCTCGGCGCGGTGCTGGTACCGCGCCTGGCGCCACGGGCCGAAGGCTCCATGGATCTACCGGCCGCA CAGCCCTTCGCCGGCCTGGCAGAGGCCGAGGCGGTACTCACCCGGGAAATCCTCGGCGCGCTGCTGG AGGCGCCGCTGGAGCTAGACGACGGTTTGCGCCGGCGCTGGCTGGACTGGCTAGCGGACTCCGCCGC CAGCGCGCTGCCGTCGCTCGACGAGGCGTTGCGCCGGCTCGGCTGGCAGGCCGCGGGGCTGACCGCG ATGGGCAACGCTCTGCGCGGCCTGCTCGCCGGCGAACAGGCGCCGGCCGCGCTGCTCCTCGATCCCT GGCTGGCGCCGCAGGCGGTGGCCGCGCGCCTGCCGGACGGCCGCGAGGCCCTGGCGCGCCTGCTCGA AGCGCTGCCGACGCCGGCTGCCGGCGAACGCCTGCGGGTGGCGGTGCTGGATACCCGCGCCGGGCTC TGGCTCGACCAGGGCATGGCCTCGCTGTTGCGCCCAGGGCTGGAACTGACCCTCTTCGAACGCAGCC GCGTCCTCCTCGACGCCGCCGCCACCCGCTTGCCGGAACGGATCGTGGTGCAGGCGCTGGACGACGG CCTGCTACCTGCCGAGCACCTCGGTCGCTACGACCGGGTGATCAGCTTCGCCGCGCTGCACGCCTAC GAGGCCAGCCGCGAAGGCCTGGCGCTGGCGGCGGCGCTGCTGCGCCCGCAGGGCCGCCTGTTGCTGG TGGACCTGCTATGCGAGTCGCCACTGGCGCTGCTCGGTGCGGCCTTGCTCGACGACCGGCCGCTGCG CCTGGCGGAGCTGCCGAGCCTGTTGGCCGATCTCGCCGCTGCGGGACTGGCGCCGCGTTGCCTGTGG CGCAGCGAGCGGATCGCCCTGGTCGAGGCGCTGGCACCGGGACTCGGGCTCGACGCCGCCGCGCTCC AGGCCGGCCTGGAGCAACGCCTGCCCCAGGCGATGCGGCCCGAACGCCTGTGGTGCCTGCCAAGCCT GCCGTTGAACGGCAATGGCAAGGTCGATCGTCGCCGCCTGGCCGAGAGCATGACCCGCGCACTCGGC GAGTGTCGTCACGAGCCCTCGGCGGAGGAGCCGCTGGAAGCCCATGAGCAAGCGCTGGCCGAGTGCT GGGAAGCGGTTCTCAAACGCCCGGTGCGTCGTCGCGAGGCGAGCTTCTTCAGCCTCGGCGGCGACAG CCTGCTGGCGACCCGCCTGCTGGCCGGCATACGTGAGCGTTTCGGCGTACGCCTGGGCATGGCCGAC TTCTATCGCCAGCCGACCCTGGCCGGTCTTGCCCGCCACTTGCAGGTGCAGACCGTCGAAATCGAGG AAACCCAACTGGAAGAGGGCGTGCTATGA

The VIR15 protein (SEQ ID NO:30) encoded by SEQ ID NO:29 is presented using the one-letter amino acid code in Table 17B. TABLE 17B Encoded VIR15 protein sequence (SEQ ID NO: 30) MDLPPDSRTALRDWLTEQLADLLGEPLADVRALADDDDLLGCGLDSIRLMYLQERLRARGSTLDFAQL AQRPCLGAWLDLLACADRLSAPATVALPTAQDRDQPFELSSVQQAYWLGRGAGEVLGNVSCHAFLEFR TRDVDPQRLAAAAECVRQRHPMLRARFLDGRQQILPTPPLSCFDLQDWRTLQVDEAERDWQALRDWRA HECLAVERGQVFLLGLVRMPGGEDRLWLSLDLLAADVESLRLLLAELGVAYLAPERLAEPPALHFADY LAHRAAQRAEAAARARDYWLERLPRLPDAPALPLACAPESIRQPRTRRLAFQLSAGESRRLERLAAQH GVTLSSVFGCAFALVLARWSESAEFLLNVPLFDRHADDPRIGEVIADFTTLLLLECRMQAGVSFAEAV KSFQRNLHGAIDHAAFPALEVLREARRQGQPRSAPVVFASNLGEEGFVPAAFRDAFGDLHDMLSQTPQ VWLDHQLYRVGDGILLAWDSVVGLFPEGLPETMFEAYVGLLQRLCDSAWGQPADLPLPWAQQARRALL NGQPACATARTLHRDFFLRAAEAPDADALLYRDQRVTRGELAERALRIAGGLREAGVRPGDAVEVSLP RGPQQVAAVFGVLAAGACYVPLDIDQPPARRRLIEEAAGVCLAITEEDDPQALPPRLDVQRLLRGPAL AAPVPLAPQASAYVIYTSGSTGVPKGVEVSHAAAINTIDALLDLLRVNASDRLLAVSALDFDLSVFDL FGGLGAGASLVLPAQEQARDAAAWAEAIQRHAVSLWNSAPALLEMALSLPASQADYRSLRAVLLSGDW VALDLPGRLRPRCAEGCRLHVLGGATEAGIWSNLQSVDTVPPHWRSIPYGRPLPGQAYRVVDTHGRDV PDLVVGELWIGGASLARGYRNDPELSARRFVHDAQGRWYRTGDRGRYWGDGTLEFLGRVDQQVKVRGQ RIELGEVEAALCAQAGVESACAAVLGGGVASLGAVLVPRLAPRAEGSMDLPAAQPFAGLAEAEAVLTR EILGALLEAPLELDDGLRRRWLDWLADSAASALPSLDEALRRLGWQAAGLTAMGNALRGLLAGEQAPA ALLLDPWLAPQAVAARLPDGREALARLLEALPTPAAGERLRVAVLDTRAGLWLDQGMASLLRPGLELT LFERSRVLLDAAATRLPERIVVQALDDGLLPAEHLGRYDRVISFAALHAYEASREGLALAAALLRPQG RLLLVDLLCESPLALLGAALLDDRPLRLAELPSLLADLAAAGLAPRCLWRSERIALVEALAPGLGLDA AALQAGLEQRLPQAMRPERLWCLPSLPLNGNGKVDRRRLAESMTRALGECRHEPSAEEPLEAHEQALA ECWEAVLKRPVRRREASFFSLGGDSLLATRLLAGIRERFGVRLGMADFYRQPTLAGLARHLQVQTVEI EETQLEEGVL MUT16

A Pseudomonas bacterial mutant (MUT16) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding pyochelin synthetase pchF (PA4225). This gene encodes the VIR16 nucleic acid (SEQ ID NO:31) shown in Table 18A. TABLE 18A VIR16 Nucleotide Sequence (SEQ ID NO: 31) ATGAGCCTCGGCGAACTGCTGGAAACCTGCCGCAGCCGGCGCATCGAACTCTGGAGCGAGGCGGGCC GCCTGCGCTATCGCGCCCCCCAGGGTGCCCTCGACGCCGGCCTCGCCGAGCGCCTGCGGGCCGAGCG CGAGGCCCTGCTGGAACACCTGGAAGGCGGCCCTGGCTGGCGCGCCGAACCCGACATGGCCCACCAG CGCTTCCCGCTGACCCCGGTGCAGGCCGCCTACGTGCTGGGCCGCCAGGCGGCCTTCGACTACGGCG GTAACGCCTGCCAGCTGTACGCCGAGTACGACTGGCCGGCCGACACCGATCCGGCGCGCCTGGAGGC GGCCTGGAACGCCATGGTCGAGCGCCACCCGATGCTGCGCGCGGTGATCGAGGACAACGCCTGGCAG CGCGTGCTGCCCGAGGTGCCCTGGCAGCGGCTGACCGTGCATGCCTGCGCGGGGCTCGACGAGGCCG CTTTCCAGGCGCACCTGGAGCGGGTCCGCGAACGCCTCGACCACGCCTGCGCGGCGCTCGACCAGTG GCCGGTCCTGCGCCCCGAGCTGAGTATCGGCCGGGATGCCTGCGTACTGCACTGCTCGGTGGATTTC ACCCTGGTCGACTACGCCAGCCTGCAATTGCTGCTTGGCGAATGGCGCCGCCGCTATCTCGATCCGC AATGGACGGCGGAACCGCTGGAGGCGACCTTCCGCGACTATGTCGGCGTCGAGCAGCGCCGACGCCA GTCGCCAGCCTGGCAGCGCGACCGCGACTGGTGGCTGGCGCGTCTCGACGCGCTACCGGGGCGTCCC GACCTGCCGCTGCGGGTGCAGCCGGACACCCGGTCCACGCGCTTCCGGCACTTCCACGCGCGCCTCG ACGAGGCCGCCTGGCAGGCGCTCGGCGCGCGCGCCGGCGAACACGGCCTGAGCGCTGCCGGCGTGGC CTTGGCGGCCTTCGCCGAGACCATCGGTCGCTGGAGCCAGGCACCGGCGTTCTGTCTCAACCTGACG GTACTCAACCGGCCGCCGCTGCATCCGCAGCTGGCGCAGGTGCTCGGTGACTTCACCGCGCTCAGCC TGCTGGCAGTGGACAGCCGCCACGGCGACAGTTTCGTCGAGCGTGCCCGACGCATCGGCGAGCAGAT GTTCGACGACCTCGACCACCCGACCTTCAGCGGCGTCGACCTGCTGCGCGAACTGGCGCGCCCGCGT GGTCGCGGCGCCGATCTCATGCCGGTGGTGTTCACCAGTGGCATCGGCAGCGTGCAGCGCCTGCTCG GCGATGGCGAGGCGCCGCGCGCGCCACGCTACATGATCAGCCAGACCCCGCAGGTCTGGCTGGACTG CCAGGTCACCGACCAGTTCGGCGGCCTGGAGATCGGCTGGGACGTACGCCTCGGGTTGTTCCCCGAG GGCCAGGCGGAAGCCATGTTCGACGACTTCGTCCGGCTGCTCCGGCGCCTGGCGCAGAGCCCGCGCG CCTGGACCGACGGCGATGCCACGGAACCCGTCGAGGCGCCGCCGCAGGCGTTGCCCGGTAGTGCCCG GAGCATCGCCGCCGGTTTCGCCGAGCGTGCCCTGCTGACCCCCGACGCCACGGCGATCCACGATGCC GCCGGCAGCTACAGCTACCGCCAGCTCGCCCAGCACGCCAGCGCCCTGCGCCGCGTCCTGGAAGCGC ACGGCGCGGGCCGTGGCCGGCGGGTCGCGGTGATGCTGCCGAAAAGCGCCGCGCAATTGGTCGCGGT GATCGGCATCCTCCAGGCCGGCGCCGCCTATGTGCCGGTGGACATCCGCCAGCCTCCGCTGCGGCGC CAGGCGATCCTCGCCAGCGCCGAAGTGGTCGCGCTGGTCTGCCTGGAAAGCGATGTCCCGGACGTCG GCTGCGCCTGCGTGGCCATCGACCGGCTGGCCGCCGACAGCGCCTGGCCGCCACCGCCCGCGGCGGA GGTGGCGGCGGACGACCTCGCCTACGTGATCTACACCTCCGGCTCCACCGGCACGCCAAAGGGCGTG ATGCTCAGCCATGCGGCGGTGAGCAACACGCTGCTCGACATCAACCAGCGCTACGGCGTCGACGCCA ACGACCGCGTCCTCGGCCTCGCCGAGCTGAGCTTCGACCTCTCGGTCTACGACTTCTTCGGCGCCAC CGCGGCGGGGGCCCAGGTGGTCCTCCCGGACCCGGCGCGCGGCAGCGATCCATCGCACTGGGCGGAA CTGCTGGAACGCCACGCCATCACCCTGTGGAACTCGGTGCCGGCCCAAGGCCAGATGCTCATCGATT ACCTGGAGAGCGAGCCGCAACGTCACCTGCCGGGACCGCGCTGCGTGCTCTGGTCCGGTGACTGGAT TCCGGTCAGCCTGCCGACCCGCTGGTGGCGGCGCTGGCCGGACAGCGCGCTGTTCAGCCTGGGCGGC GCCACCGAGGCGGCGATCTGGTCGATCGAGCAGCCGATCCGCCCGCAGCACACCGAGCTGGCCAGCA TCCCTTATGGCCGTGCCCTGCGCGGGCAGAGCGTGGAAGTCCTGGATGCCCGCGGGCGGCGCTGCCC GCCGGGCGTGCGCGGCGAGATCCATATCGGCGGGGTGGGCCTGGCGCTCGGCTACGCCGGCGATCCG CAGCGCACCGCCGAACGCTTCGTCCGTCACCCCGATGGCCGTCGCCTGTATCGCACCGGCGACCTCG GCCGCTACCTGGCCGACGGCAGCATCGAGTTCCTCGGCCGCGAGGACGACCAGGTGAAGATTCGCGG CCACCGCATCGAACTGGCCGAACTGGACGCCGCGCTGTGCGCTCATCCGCAGGTCAACCTGGCGGCC ACCGTGGTGCTCGGCGAGACCCACGAGCGCAGCCTGGCCAGCTTCGTCACCCTGCATGCGCCGGTGG AGGCTGGCGAGGATCCGCGTACGGCGCTCGACGCGGTGCGCCAGCGGGCGGCCCAGGCCTTGCGCCG CGACTGGGGCAGCGAGGAGGGCATCGCCGCGGCGGTGGCCGCACTCGACCGTGCCTGCCTCGCCTCG TTGGCCGCCTGGCTGGCCGGCAGCGGTCTGTTCGCCAGTGCGACGCCGCTGGACTTAGCCACCCTGT GCCAGCGCCTGGGTATCGCCGAGGCGCGCCAGCGCCTGCTGCGCCACTGGTTGCGCCAACTGGAGGA GGGCGGCTACCTGCGCGCCGAGGGCGAGGGCTGGCTGGGCTGCGCCGAGCGTCCCGCGCAGAGTCCG GAGGACGCCTGGACGGCGTTCGCCGGCTGCGCGCCGGCGGCGCTCTGGCCGGCCGAGCTGGTCGCCT ACCTGCGTGACAGCGCGCAATCCCTCGGCGAGCAACTGGCCGGGCGGATCAGCCCGGCGGCGCTGAT GTTCCCGCAGGGCTCGGCGCGCATCGCCGAGGCCATGTACAGCCAGGGCCTGCATGCCCAGGCGCTG CACGAGGCCATGGCCGAGGCCATCGCCGCCATCGTCGAGCGCCAGCCGCAACGGCGCTGGCGCCTGC TGGAGCTTGGCGCCGGCACCGCCGCCGCCAGCCGCACGGTGATCGCCCGGTTGGCGCCGCTGGTGCA GCGAGGGGCGGAGGTGGACTACCTGTTCACCGACGTTTCCAGCTACTTCCTCGCCGCCGCCCGCGAG CGCTTCGCCGACCAGCCGTGGGTACGCTTCGGCCGCTTCGACATGAACGGCGATCTTCTCGACCAGG GCGTGGCGCCGCACTCGGTGGATATCCTGCTCAGCTCCGGGGCCTTGAACAACGCGCTGGACACCCC GGCGCTGCTCGCCGGCCTGCGCGAGTTGCTGAGCGCCGACGCCTGGCTGGTGATCCAGGAACTGACG CGCGAGCACAACGAGATCAGCGTCAGCCAGAGCCTGATGATGGAAAACCCGCGCGACCTCCGCGACG AGCGCCGCCAACTGTTCGTCCACACCGGGCAATGGCTGGAGTGGCTGGCGGCACAGGGTGGCGACCT GGCTTGTGGGGTGGTGCCGCCGGGCAGCGCTCTCGACCTGCTTGGCTACGATGTCCTGCTGGCTCGC TGCAAGACCGACCGCGCCCGCCTGGAGCCGGCCGAGCTGCTGGCCTTCGTCGAAGCGCGGGTGCCGC GCTACATGCTCCCGGCGCAGTTGCGCGTGCTCGAACGCCTGCCGGTCACCGGCAACGGCAAGATCGA CCGCAAGGCCCTGACCGGCTTTGCCCGCCAGCCCCAGGCGGACCTTCGGCATGGCGTCGCGCAGGCA CCGGCCGACGAACTGGAGAATGCGCTGCTGGCACTCTGGCGGGAGGTGCTGGACAACCCGTCGCTGG GCGTCGAGCAAGACTTCTTCGGGGCCGGCGGCGACTCGCTGTTGATCGCCCAGTTGATCGCCCGTTT GCGCGAACGACTGGAAAGCGCCCGTCGGCATCCGTTCGATCGCCTGCTACGCTGGGCGCTCAGCCAG CCGACGCCGCGCGGCCTGGCCGAACGCCTGCGCAGCGCGCCGGAAGAGGGCCGTGGGCCAGCCCTGG CCGCGGCGCGCGGCGTCGCCCCGGCGCCGGCCGGCATGTCGCGCGCACCGCTCGCCGAGGGCGCGGT GGCGCTCGACCCGCTGGTGCGCCTGGTGCCCGGCGAGGGCGTGCCGCGGGTGCTGGTCCACGAAGGC CTCGGCACGCTACTGCCGTACCGCCCGCTGCTTCGCGCCCTGGGTGAGGGGCGGCCGTTGCTGGGGC TGGCCGTGCATGACAGCGACGCCTACCTGGCGATCCCCGCCGAGCATCTCAACGCCTGCCTCGGCCG CCGCTACGCCGAGGCGCTCCATCGCGCCGGGCTACGCGAGGTCGACCTGCTCGGCTACTGCTCCGGC GGGCTGGTCGCCCTGGAGACCGCCAAGTCCCTGGTCCAGCGCGGGGTGCGCGTGCGCCAACTGGATA TCGTCTCCAGCTACCGGATTCCCTACCGGGTGGACGACGAGCGCCTGCTGTTGTTCAGCTTCGCCGC GACCCTCGGCCTGGATACCGCGGCGCTCGGCTTCCCCGCGCCGGAACGTCTCGGCCAGGCGGTGCAG GCGGCGCTCGCGCAGACACCGGAGCGCCTGGTCGCCGAGGCGCTGGCGGGGCTGCCGGGCCTGGCCG ATCTCGTCGCCCTGCGCGGCCGCGTGCTACAGGCGGCCAGCGGTAGCGCCGACGCCGTCAGCGTCGA ACGCGACACCCTCTACCGGCTGTTCTGTCACTCGGTGCGTGCCAGCCAGGCCGAGGCGCCGGAGCCC TACGTCGGCGCGCTGCGGCTGTTCGTGCCGGACGCCGGCAACCCATTGGTGCCGCGCTACGCCGAGG CTCTGGAGACCCAATGGCGGGCCGCCGCGCTTGGCGCGTGCGGCATCCACGAGGTGCCCGGCGGGCA CTTCGACTGCCTGGGCGAAGCCCTGGCGCAATCCTTGTCGAAACCCATGCCAGAGGAGGCGAGCCGA TGA

The VIR16 protein (SEQ ID NO:32) encoded by SEQ ID NO:31 is presented using the one-letter amino acid code in Table 18B. TABLE 18B Encoded VIR16 protein sequence (SEQ ID NO: 32) MSLGELLETCRSRRIELWSEAGRLRYRAPQGALDAGLAERLRAEREALLEHLEGGPGWRAEPDMA HQRFPLTPVQAAYVLGRQAAFDYGGNACQLYAEYDWPADTDPARLEAAWNAMVERHPMLRAVIED NAWQRVLPEVPWQRLTVHACAGLDEAAFQAHLERVRERLDHACAALDQWPVLRPELSIGRDACVL HCSVDFTLVDYASLQLLLGEWRRRYLDPQWTAEPLEATFRDYVGVEQRRRQSPAWQRDRDWWLAR LDALPGRPDLPLRVQPDTRSTRFRHFHARLDEAAWQALGARAGEHGLSAAGVALAAFAETIGRWS QAPAFCLNLTVLNRPPLHPQLAQVLGDFTALSLLAVDSRHGDSFVERARRIGEQMFDDLDHPTFS GVDLLRELARRRGRGADLMPVVFTSGIGSVQRLLGDGEAPRAPRYMISQTPQVWLDCQVTDQFGG LEIGWDVRLGLFPEGQAEAMFDDFVGLLRRLAQSPRAWTDGDATEPVEAPPQALPGSARSIAAGF AERALLTPDATAIHDAAGSYSYRQVAQHASALRRVLEAHGAGRGRRVAVMLPKSAAQLVAVIGIL QAGAAYVPVDIRQPPLRRQAILASAEVVALVCLESDVPDVGCACVAIDRLAADSAWPPPPAAEVA ADDLAYVIYTSGSTGTPKGVMLSHAAVSNTLLDINQRYGVDANDRVLGLAELSFDLSVYDFFGAT AAGAQVVLPDPARGSDPSHWAELLERHAITLWNSVPAQGQMLIDYLESEPQRHLPGPRCVLWSGD WIPVSLPTRWWRRWPDSALFSLGGATEAAIWSIEQPIRPQHTELASIPYGRALRGQSVEVLDARG RRCPPGVRGEIHIGGVGLALGYAGDPQRTAERFVRHPDGRRLYRTGDLGRYLADGSIEFLGREDD QVKIRGHRIELAELDAALCAHPQVNLAATVVLGETHERSLASFVTLHAPVEAGEDPRTALDAVRQ RAAQALRRDWGSEEGIAAAVAALDRACLASLAAWLAGSGLFASATPLDLATLCQRLGIAEARQRL LRHWLRQLEEGGYLRAEGEGWLGCAERPAQSPEDAWTAFAGCAPAALWPAELVAYLRDSAQSLGE QLAGRISPAALMFPQGSARIAEAMYSQGLHAQALHEAMAEAIAAIVERQPQRRWRLLELGAGTAA ASRTVIARLAPLVQRGAEVDYLFTDVSSYFLAAARERFADQPWVRFGRFDMNGDLLDQGVAPHSV DILLSSGALNNALDTPALLAGLRELLSADAWLVIQELTREHNEISVSQSLMMENPRDLRDERRQL FVHTGQWLEWLAAQGGDLACGVVPPGSALDLLGYDVLLARCKTDRARLEPAELLAFVEARVPRYM LPAQLRVLERLPVTGNGKIDRKALTGFARQPQADLRHGVAQAPADELENALLALWREVLDNPSLG VEQDFFGAGGDSLLIAQLIARLRERLESARRHPFDRLLRWALSQPTPRGLAERLRSAPEEGRGPA LAAARGVAPAPAGMSRAPLAEGAVALDPLVRLVPGEGVPRVLVHEGLGTLLPYRPLLRALGEGRP LLGLAVHDSDAYLAIPAEHLNACLGRRYAEALHRAGLREVDLLGYCSGGLVALETAKSLVQRGVR VRQLDIVSSYRIPYRVDDERLLLFSFAATLGLDTAALGFPAPERLGQAVQAALAQTPERLVAEAL AGLPGLADLVALRGRVLQAASGSADAVSVERDTLYRLFCHSVRASQAEAPEPYVGALRLFVPDAG NPLVPRYAEALETQWRAAALGACGIHEVPGGHFDCLGEALAQSLSKPMPEEASR MUT17

A Pseudomonas bacterial mutant (MUT17) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding, putative ATP-binding component of the ABC transporter, pchH (PA4223). This gene encodes the VIR17 nucleic acid (SEQ ID NO:33) shown in Table 19A. TABLE 19A VIR17 Nucleotide Sequence (SEQ ID NO: 33) GTGACCCCGGTGCTGTGGCGCCTGCTGCGCACCTATCGCTGGCGGCTGGCGGCGGCCATGGGGTTGC AGGCCCTGGCCGGGCTCTGCTCGCTGTTGCCCTGGATGCTTCTCGCCTGGCTCGCCGAGCCGCTGGC GCGCGGCCAGGCGCAGCCGGCCCTGCTGGCCCTGGTGCTGCTGGCGGTGCTGGCCTGGCTGGGCTGC CAGGCGCTGCCCGCGCACCTGGCCCACCGGGTCGACGCGGACCTCTGCAACGACCTGCGCCTGCGCC TGCTGGCGCACCTGCAACGGCTGCCGCTGGACTGGTTCGGTCGCCAGGGCCCGGACGGCGTGGCGCG CCTCGTGGAGCAGGACGTGCGGGCCCTGCACCAACTGATCGCGCACGCTCCCAACGATCTCAGCAAC CTGTTGGTGGTGCCGCTCGTCGCGTTGCTCTGGCTGGCCTGGCTGCACCCCTGGCTGCTGCTGTTCT GCCTGCTGCCGCTGGTGCTGGCCGCCGCCGGCTTCCTGCTGCTGCGCTCGGCGCGCTACCGCGACCT GGTGCTGCGGCGCAACGCCGCGCTGGAAAGGCTCTCGGCGGACTATGGCGAATTCGCCCACAACCTG CTGCTGGCCCGACAGTACCCCGGCGCCGGCATACAACAGGGCGCCGAGGCGTCGGCGGCGGCCTTCG GCGAAGCGTTCGGCGCCTGGGTGAAGCGGGTCGGCCACCTCGCCGCGCTGGTCTACGTGCAGTTGTC GACGCCCTGGCTGCTGGCCTGGGTCCTGCTCGGCGCGCTGGCCCTGGATGCCCTCGGCGTGCCGCTG GCGCTCGGCCAGGCCTGTGCCTTCCTGCTCCTGCTGCGGGCCTTGGCTGCCCCGGTGCAGGCGCTCG GCCACGCCGGCGACGCGCTGCTGGGCGCGCGCGCCGCCGCCGAGCGCCTGCAGCAGGTGTTCGACCA GGCGCCGCTGGCCGAGGGCCGCTCGACCCGCGAGCCGGTCGATGGCGCGGTGGCGCTGCACGGCCTG GGCCATGCCTATGAAGGCGTGGAGGTCCTGGCCGATATCGATCTGGAGCTGGAGGATGGCAGCCTGG TGGCCCTGGTCGGTCCCTCGGGCTCCGGCAAGAGCACCCTGCTGCACCTGCTGGCGCGCTACATGGA CGCGCAGCGCGGCGAACTGGAGGTTGGCGGCCTGGCACTGAAGGACATGCCTGATGCCGTGCGCCAT CGGCATATCGCGCTGGTCGGCCAGCAGGCGGCGGCGCTGGAGATATCCCTGGCCGACAACATTGCCC TGTTCCGCCCCGATGCCGATCTCCAGGAGATTCGCCAGGCGGCCCGTGACGCCTGCCTCGACGAGCG CATCATGGCCCTGCCGCGTGGCTACGACAGCGTGCCGGGACGCGACCTGCAACTGTCCGGCGGCGAA CTGCAACGACTGGCCCTGGCCCGTGCGCTGCTATCGCCGGCGAGCCTGTTGCTGCTCGACGAGCCAA CCTCGGCGCTGGATCCGCAGACCGCCCGGCAGGTCCTGCGCAACCTGCGCGAACGCGGCGGTGGCCG GACCCGGGTGATCGTCGCCATCGTCTGGCCGAAGTCAGCCGATGCCGACCTGATCCTGGTGCTGGTC GCTGGCCGTCTGGTCGAACGCGGCGAGCACGCGGCGCTGTTGGCGGCGGACGGCGCCTATGCGCGCT TGTGGCGTGAACAGAACGGCGCGGAGGTGGCGGCATGA

The VIR17 protein (SEQ ID NO:34) encoded by SEQ ID NO:33 is presented using the one-letter amino acid code in Table 19B. TABLE 19B Encoded VIR10 protein sequence (SEQ ID NO: 34) MTPVLWRLLRTYRWRLAAAMGLQALAGLCSLLPWMLLAWLAEPLARGQAQPALLALVLLAVLAWL GCQALAAHLAHRVDADLCNDLRLRLLAHLQRLPLDWFGRQGPDGVARLVEQDVRALHQLIAHAPN DLSNLLVVPLVALLWLAWLHPWLLLFCLLPLVLAAAGFLLLRSARYRDLVLRRNAALERLSADYG EFAHNLLLARQYPGAGIQQGAEASAAAFGEAFGAWVKRVGHLAALVYVQLSTPWLLAWVLLGALA LDALGVPLALGQACAFLLLLRALAAPVQALGHGGDALLGARAAAERLQQVFDQAPLAEGRSTREP VDGAVALHGLGHAYEGVEVLADIDLELEDGSLVALVGPSGSGKSTLLHLLARYMDAQRGELEVGG LALKDMPDAVRHRHIALVGQQAAALEISLADNIALFRPDADLQEIRQAARDACLDERIMALPRGY DSVPGRDLQLSGGELQRLALARALLSPASLLLLDEPTSALDPQTARQVLRNLRERGGGRTRVIVA HRLAEVSDADLILVLVAGRLVERGEHAALLAADGAYARLWREQNGAEVAA

The role of VIR17 in virulence was confirmed using phage to retransduce this mutation into the wild-type PT894 strain where attenuated virulence was again observed in the Dictyostelium growth assay compared to an isogenic bacterial strain.

MUT18

A Pseudomonas bacterial mutant (MUT18) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding the putative ATP-binding component of ABC transporter, pchI (PA4222). This gene encodes the VIR18 nucleic acid (SEQ ID NO:35) shown in Table 20A. TABLE 20A VIR18 Nucleotide Sequence (SEQ ID NO: 35) ATGACCCTGTTCGAACGAATGCGTGCGCTGCCCGAAGACTGCCGTGCCGCGTTGCGCCGGGCGAGCG CCTGGGCGGTCCTGGCGGCGCTGCTGGACGCCGCTTGCGGCGTATTGCTGGTGCCGTTGGTCGAGGC CTGGTTCGCCGAAGGCGCGTTGCCCTGGCGCTGGGTCGCCGCGTTGCTCGGCTTGAGCCTGGCGCAG GCGCTGTTGCAGTACCTGGCCCTGCGTCGCGGTTTCGCCGCCGGCGGCTCGCTGGCGGCTGGACTGG TGCGCAGCCTGGTGGCGCGCTTGCCGCGCCTGGCGCCGCCGGCGCTGCGCCGGGTCGCGCCGGCCGA AGGCCTGCTGCGCGGCCCGGTGATGCAGGCGATGGGCATTCCGGCGCACCTGCTGGGGCCGCTGATC GCCGCGTTGGTGACGCCGCTCGGGGTGATCCTCGGGCTGTTCCTGATCGACCCGTCCATCGCCCTCG GCCTGCTCCTTGCTGGTGCCTTCCTCGCCGCGCTGTTGCGCTGGAGCGGGCGGCGCAATCTGGCGGC GGAGGATGCCCGGCTGGCCGCCGAGCGCGACGCCGCACGGCAGTTGCAGGCGTTCGCCGAACGCCAG CCACTGCTGCGCGCGGCGCAGCGCGAAAGCGTCGCCCGCCAGGGGCTGGAAGAGGCCTTGCGCAGTC TCCACCGCAGCACCCTGGATCTGTTGCGGCGCAGCCTGCCCAGCGGCCTCGGCTTCGCCCTGGCGGT GCAGGCGGCGTTCGCCTTCGCCCTGCTCGGCGGCGCCTGGGCGGTGGAGCGGCAATGGCTGGACCGC GCTCGGCTGGTGGCCGTGCTGGTGCTGCTGGTGCGCTTCATCGAGCCGCTGGCCCAGCTCACCCATC TCGACCAGGCGTTGCGCGGCGCCTGGCAGGCGCTGGATACCCTGCTGCGGGTTTTCGCCCTGGCTCC GCTGCGCAGCCCCGAGCCGGGCGAGCGGCCGCACGACGCCAGCCTGGCGGCCGAGGCCGTGGAATTG CGCCTGGAAGATGGCCGCGCCTTGCTCGAGGACATTTCCCTGAGGCTGGAGCCGGGTTCGCTGAACG TCCTCGTCGGACCCTCCGGGGCCGGCAAGAGCAGCCTGCTGGCGCTGCTCGGGCGGCTCTACGACGT CGATGCCGGGCGTGTCCTGCTGGGTGGCGTGGATATCCGCCGGTTGAGCGAAACGACCCTCGCCGCC AGTCGTAACCTGGTGTTCCAGGACAACGGCCTGTTCCGCGGCAGCGTTGCCTGGAACCTGCGCATGG CGCGAGCGGACGCCGATCTCGAAGCGCTGCGCGAGGCGGCGCGGGCGGTTGGCCTGCTGGAAGAGAT CGAGGCCTGGCCGCAGGGCTGGGACAGCGACGTCGGTCCCGGCGGCGCGCTGCTGTCCGGCGGCCAG CGGCAACGCCTGTGCCTGGCTCGCGGGCTGCTCTCGACGGCGCCGTTGCTGCTGCTCGACGAGCCCA CCGCCAGCCTCGACGCCGCCAGCGAGGCGCAGGTGCTGCGCAGCCTGCTCGGGTTGCGCGGCCGGCG CACCCTGCTGGTAGTGACCCACCGCCCGGCGCTGGCGCGTCAGGCCGACCAGGTACTGCTGCTGGAG GAGGGGCGCCTGCGCCTCACCGGACTTCACGCCGATCTGCTCGTCCGGGACGACTGGTATGCCGGTT TCGTCGGGCTGGCGGGCGAGGAAAGTTCCGCGACGGTCGTGGATCGATAG

The VIR18 protein (SEQ ID NO:36) encoded by SEQ ID NO:37 is presented using the one-letter amino acid code in Table 20B. TABLE 20B Encoded VIR18 protein sequence (SEQ ID NO: 36) MTLFERMRALPEDCRAALRRASAWAVLAALLDAACGVLLVPLVEAWFAEGALPWRWVAALLGLSL AQALLQYLALRRGFAAGGSLAAGLVRSLVARLPRLAPPALRRVAPAEGLLRGPVMQAMGIPAHLL GPLIAALVTPLGVILGLFLIDPSIALGLLLAGAFLAALLRWSGRRNLAAEDARLAAERDAARQLQ AFAERQPLLRAAQRESVARQGLEEALRSLHRSTLDLLRRSLPSGLGFALAVQAAFAFALLGGAWA VERQWLDGARLVAVLVLLVRFIEPLAQLTHLDQALRGAWQALDTLLRVFALAPLRSPEPGERPHD ASLAAEAVELRLEDGRALLEDISLRLEPGSLNVLVGPSGAGKSSLLALLGRLYDVDAGRVLLGGV DIRRLSETTLAASRNLVFQDNGLFRGSVAWNLRMARADADLEALREAARAVGLLEEIEAWPQGWD SDVGPGGALLSGGQRQRLCLARGLLSTAPLLLLDEPTASLDAASEAQVLRSLLGLRGRRTLLVVT HRPALARQADQVLLLEEGRLRLSGLHADLLVRDDWYAGFVGLAGEESSATVVDR

The role of VIR18 in virulence was confirmed using phage to retransduce this mutation into the wild-type PT894 strain where attenuated virulence was again observed in the Dictyostelium growth assay compared to an isogenic bacterial strain.

MUT19

A Pseudomonas bacterial mutant (MUT19) was made by transposon insertion in a P. aeruginosa wild-type strain PT894. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as a gene cluster encoding the P. aeruginosa serotype 09 putative O-antigen biosynthesis pathway (VIR19). The insertion site nucleic acid sequence identifying the VIR19 gene in MUT19 is shown in Table 21. TABLE 21 MUT19 Transposon insertion Site (SEQ ID NO: 37) CTCTTTCAGCCGCACGCGGCGCACCTCGTGTGTGATCAGTGAGTGGTTTGCAACTGCGGGTCAAG GATCTGGATTTCCCTCACANGTNCGATCATCGTGCGGGAGGGCAAGGGCTCCAAGGATCGGGCCT TGATGTTACCCGAGAGCTTGGCACCCAGCCTGCGCGAGCAGGGNNAATTGATCCGGTGGATGACC TTTTGAATGACCTTTAATAGATTATATTACTAATTAATTGGGGACCCTANAGGTCCCCTTTTTTA TTTTAAAAATTTTTTCACAAAACGGTTTATTTNCATAAAGCTTGCTCAATCAATCACCNTATCCN CGGGAATTCGGCCTAGGCGGCCAGATCTGATCAAGAGACAGACCTCCAGCTTTGCATCCGGAGCG ACCACACGAGCGAGGTCAGTCACTTTCATCGAAGGAATTTTCTTGACATAGATCTCACCACCTTC CATGTCCTCAAAGGCATGCCACACTAACTCGACGCCCTCCTCCAAAGAAATCATGAACCGGGTCA TCCGCTCATCAGTGATAGGCAAGACGCCCTTGTCCTTG

The role of this cluster in virulence was confirmed using phage to retransduce this mutation into the wild-type PT894 strain where attenuated virulence was again observed in the Dictyostelium growth assay compared to an isogenic bacterial strain.

B. Attenuated Klebsiella Mutants

MUT20

A Klebsiella bacterial mutant (MUT20) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding a hypothetical transcriptional regulator in met G-dld intergenic region (VIR20). The insertion site nucleic acid sequence identifying the VIR20 gene in MUT20 is shown in Table 22. TABLE 22 MUT20 Transposon Insertion Site (SEQ ID NO: 38) ACGCAGGATATCTTCTTCATCAAATTGTCGATGCCCGCCTTCGCTACGCTGCGGTTTCAGTAGACCG TAACGACGCTGCCAGGCGCGCAGTGTGACCGGATTGATTCCGCAACGTTCGGCGACTTCACCGATAC TGTAAAACGCCATAGCAGCCTCACATCAACCTGATACCTTAATACCTAAACTAACGAATTCAGGCAT CCTGTACAACTCTATTTTCTTGTACAGATAAAGATATCAGGTTGCGGCTCACAGCGCCCGGGAAAAA AGATGAAAAAATGTTTAGCTGATTTCGCGGTGGTTCATTTTTTCTCCGGCCATGCGACGGCGGGTAG GCCCCCCAGGCGCGCGCTGGCGAACAAATTGCCCTGAAACTGTGAAATACCGGCTGATTCCAGCCAC ATCCACTCTTCAGCACGCTCAACGCCGACGGCTGAGACCGCAATCTCCAGAGAAGTACAGCATTTGA TAATCGCCTG MUT21

A Klebsiella bacterial mutant (MUT21) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding •-cystathionase (VIR21). The insertion site nucleic acid sequence identifying the VIR21 gene in MUT21 is shown in Table 23. TABLE 23 MUT21 Transposon Insertion Site (SEQ ID NO: 39) GACCATGTGCTGATGACCAATACCGCCTATGAGCCAAGCCAGGACTTTTGTACCAAAATTCTCGCCA AACTCGGCGTCACCACCAGCTGGTTCGATCCCTTAATCGGCGCCGATATCGCCCGTCTGGTTCGCCC TGAGACCCGCGTGGTGTTCCTCGAATCGCCCGGCTCGATCACCATGGAAGTGCACGATGTGCCGGCG ATAGTCGCCGCCGTGCGTCAGGTCGCCCCGGAAGCGATTATCATGATCGATAACACCTGGGCGGCGG GGATCCTGTTTAAAGCCCTGGATTTTGGCATTGATATTTCCATTCAGGCAGGCACCAAATACCTGAT CGGCCATTCCGACGCCATGGTGGGCACCGCGGTGGCGAACGCGCGCTGCTGGCCGCAGCTGCGTGAA AATGCCTACCTGATGGGGCAAATGCTGGACGCCGATACTGCCTATATGACCAGCCGCGGCCTGCGAA CCCTGGGCGTGCGCCTGCGTCAGCATCATGAAAGCAGCCTGCGCATC MUT22

A Klebsiella bacterial mutant (MUT22) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as ribosome binding factor A (VIR22). The insertion site nucleic acid sequence identifying the VIR22 gene in MUT22 is shown in Table 24. TABLE 24 MUT22 Transposon Insertion Site (SEQ ID NO: 40) CTTTTGGCCCCTTTTTTGTCTTTATTCTGGAGAACTTATTATGGCGAAAGAATTTGGTCGCCCGCAG CGTGTGGCCCAGGAGATGCAAAAAGAGATTGCCATCATCCTGCAGCGTGAAATTAAAGATCCGCGTC TGGGCATGATGACCACCGTTTCCGGTGTGGAAATGTCCCGTGACCTGGCCTATGCCAAGGTGTATGT CACCTTCCTTAACGACAAAGATGAAGCCGCGGTGAAAGCGGGCATCAAAGCGCTGCAGGAAGCTTCT GGCTTTATCCGCTCTCTGCTGGGGAAAGCGATGCGTCTGCGCATCGTACCGGAACTGACTTTCTTCT ACGACAACTCACTGGTGGAAGGGATGCGTATGTCCAACCTGG MUT23

A Klebsiella bacterial mutant (MUT23) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding aspartokinase/homoserine dehydrogenase (VIR23). The insertion site nucleic acid sequence identifying the VIR23 gene in MUT23 is shown in Table 25. TABLE 25 MUT23 Transposon Insertion Site (SEQ ID NO: 41) GCCCAGCCCGCTTTCCCGCTTGCCCAGTTAAAAGCCTTCGTGGAGCAGGAATTTGCTCAGATTAAGC ATGTTCTGCACGGCATCAGCCTGCTGGGTCAGTGCCCGGACAGCGTCAATGCCGCGCTGATCTGCCG CGGCGAAAAGCTCTCCATCGCCATCATGGCGGGTCTGCTGGAAGCCCGTGGACACAAAGTCAGTGTC ATTAACCCGGTCGAAAAACTGCTCGCCGTGGGTCACTATCTGGAATCCACCGTCGATATCGCCGAAT CCACCCGCCGCATTGCCGCCAGCCAGATCCCGGCAGACCATATGATCCTGATGGCCGGGTTTACCGC CGGCAATGAGAAAGGCGAGCTGGTGGTGCTGGGGCGTAACGGCTCCGACTACTCGGCTGCGGTACTG GCCGCCTGCCTGCGCGCTGACTGCTGCGAAATCTGGACCGATGTCGACGGAGTGTACACCTGCGATC CGCGTCAGGTGCCGGATGCGCGCCTGCTGAAATCGATGTCTTATCAGGAGGCGATGGAGCTCTCCTA CTTTGGCGCGAAAGTGCTGCACCCGCGCACCATTGCCCCTATCGCCCAGTTCCAAATCCCATGCCTG ATTAAAAATACCGGCAACCCCC MUT24

A Klebsiella bacterial mutant (MUT24) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding cystathione •-synthetase (VIR24). The insertion site nucleic acid sequence identifying the VIR24 gene in MUT24 is shown in Table 26. TABLE 26 MUT24 Transposon Insertion Site (SEQ ID NO: 42) GGCGCAGCGTCTGCTCGTCACCGTCAAGCTCGAAGCTTAACATTGCGCCAAAACCTTTTTGCTGACG CGCCGCAATTTCATGCCCCTGGTTTTCCGGCAGCGATGGATGATACAGCTTTTTCACCAGCGGCTGG GTTTTCAGATACTCAACGATCGCCAGGGCATTTCGCTGCGCCACTTCCATCCGTGGAGACAGCGTCC GCAGCCCGCGCAACAGCAGATAGCTGTCGAAGGCGCTGCCGGTGACGCCAATATTATTCGCCCACCA TGCCAGTTCGGTGACAGTTGCCGGATCTTTGGCAATCACCACCCCGGCCACCACATCGGAGTGACCA TTGAGGTATTTGGTACAGGA MUT25

A Klebsiella bacterial mutant (MUT25) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding phosphoribosylformylglycinamidine synthase (VIR25). The insertion site nucleic acid sequence identifying the VIR25 gene in MUT25 is shown in Table 27. TABLE 27 MUT25 Transposon Insertion Site (SEQ ID NO: 43) GTTGCGTCCCAGGCGCGTAAACGCATCCTGCAGGTAGTCAATTTCGTCGTCGGCCAGCGCCAGACCC AGACGGAGGTTGGCGTCAATCAGCGCCTGACGCCCTTCGCCCAGCAGGTCGACGCTGGTGACCGGCG TCGGCTGATGGTGAGCGAACAGCTTCTCGCCCGCTTCCAGCTCGTCGAAGACGCTCTCCATCATGCG GTCATGCAGCTCCGCCGCCACCGCGGCCCACTGCGCTTCGGTCAGGGTTGAGGCTTCAACGTAATAC GCCACGCCGCGCTCAAGACGCACAACCTGCGCCAGACCGCAGTTGTGAGCGATATCGGTAGCTTTAG AAGACCAGGGAGAGATGGTGCCAGGGCGAGGGGTCACGAGCAGTAATTTACCGGTCGGGGTATGGCT GCTTAAGCTCGGGCCATACTGAAGCAGTCGCGCCAGGCGCTCGCGATCGTCAGCGCTCAGCGGGCCG TTCAGATCGGCAAAATGAATATATTCGGCAT MUT26

A Klebsiella bacterial mutant (MUT26) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding homoserine transsuccinylase (VIR26). The insertion site nucleic acid sequence identifying the VIR26 gene in MUT26 is shown in Table 28. TABLE 28 MUT26 Transposon Insertion Site (SEQ ID NO: 44) GTATTGGCATCGTACTCCTGGGCTGGCCGGTGACAAAGGCGATGCGCTTATCTTTGCTGGCGAACAA ATACGCATCGCCCTCTTCCGTCTCCGCGAGGATCTCGAGATCGGTATAGTCGCGAATAAGTCCGGCC GGAAAATCAGCATAGCGTGAGTGCGGGGCCAGGAAAGAGTCGTCGAAACCGCGGGTCAGTAAGGCGT GCGGATGAAGAATATGGTGTTCATAGACGCCGGAAATCTTTTCGGCGCGGGTCTGCTTGGGAATGCC GTACAGAATGTTCAGCGCGGCCTGAACCGCCCAACAGACGAACAGCGTCGAAGTGACGTGATCCTTG GCCCACTCCAGCACCTGTTTGATCTGCGGCCAGTAAGCAACATCGTTAAACTCAACCAGGCCTAAAG GAGCGCCGGTAACAATCAGGCCGTCAAAGTTCTGATC MUT27

A Klebsiella bacterial mutant (MUT27) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding 3′-phosphoadenosine 5′-phosphosulfate reductase (VIR27). The insertion site nucleic acid sequence identifying the VIR27 gene in MUT27 is shown in Table 29. TABLE 29 MUT27 Transposon Insertion Site (SEQ ID NO: 45) GAGGTTCATATGTCCGTACTCGATCTAAACGCGCTTAATGCATTGCCGAAAGTGGAACGCATTCTGG CACTCGCGGAAACCAACGCCCAACTGGAAAAGCTTGACGCCGAAGGGCGTGTGGCGTGGGCGCTGGA AAATCTGCCGGGAAACTATGTGCTGTCGTCGAGCTTTGGCATTCAGGCGGCGGTAAGTTTGCATCTG GTGAATCAGATCCGCCCGGACATTCCGGTGATCCTCACCGATACCGGCTACCTGTTCCCGGAAACCT ATCAGTTTATTGACGAGCTGACGGACAAG MUT28

A Klebsiella bacterial mutant (MUT28) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding Sfi protein (VIR28). The insertion site nucleic acid sequence identifying the VIR28 gene in MUT28 is shown in Table 30. TABLE 30 MUT28 Transposon Insertion Site (SEQ ID NO: 46) TGTTAAAGCGTGCGTTCTACAGCCTGTTAGTCCTGCTCGGCCTGCTGCTGTTGACCGTGCTGGGCCT TGACCGCTGGATGAGCTGGAAAACCGCGCCCTATATCTATGATGAACTGCAGGACCTGCCCTACCGT CAGGTCGGTGTGGTGCTGGGCACCGCCAAATATTACCGCACCGGCGTCATCAATCAGTATTACCGTT ACCGCATCCAGGGTGCGCTGAACGCCTACAACAGCGGCAAGGTCAACTATCTCCTGCTGAGCGGCGA TAATGCTCTGCAAAGCTACAATGAACCGATGACCATGCGTCGGGACCTGATTAAAGGCGGCGTCGAT CCCGCGGATATCGTACTGGACTATGCCGGTTTCCGTACCCTCGACTCGATCGTCCGTACCCGGAAAG TGTTCGACACCAACGACTTCATTATCATCACCCAGCGCTTCCACTGCGAACGGGCGCTGTTTATCGC CCTGCATATGGGGATCCAGGCCCAGTGCTACGC MUT29

A Klebsiella bacterial mutant (MUT29) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding transcriptional activator protein LysR (VIR29). The insertion site nucleic acid sequence identifying the VIR29 gene in MUT29 is shown in Table 31. TABLE 31 MUT29 Transposon Insertion Site (SEQ ID NO: 47) CGCTGAACCTCCTCAAACAAACGCAGGCCCTGCACCTGTCGGCTGCAGGCGACCAGCGTGGATCCGC TCAAACAGCTGCAGGCCGAGCACCTTCTCAAAGCGCGCCAGCTCGCGGCTGACCGTGGGTTGCGAGG TGTGCAGCATCCGCGCCGCTTCGGTCAGGTTGCCGGTGGTCATCACCGCGTGAAAGATTTCGATATG ACGCAAATTGACGGCTGGCATGCGGTCTCCGTGAGGCTCGGCTGGAACCATATCATTTTTGCATAGA GTCGCGATAAAACGATATTTTTTATTCGTCTGTCACTGTGGCGTAATCAGAAAAAACAGCGACCAAC ACACGCACTGCACCGGAGTTCTTATGCCACACTCGCTTTACGCCACCGATACTGACCTGACCGCGGA CAACCTGCTGCGCCTGCCGGCGGAATTTGGCTGCCCGGTCTGGGTCTATGATGCGCAGATTATTCGC CGCCAGATAGCCCAGCTCAGCCAGTTTCGAC MUT30

A Klebsiella bacterial mutant (MUT30) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding TrpD (VIR30). The insertion site nucleic acid sequence identifying the VIR30 gene in MUT30 is shown in Table 32. TABLE 32 MUT30 Transposon Insertion Site (SEQ ID NO: 48) GGCTTCCACCCAAATCGCTTTGTCGGCAACGATTTTTGCTAAAACGGCTTTGCATTCTTTACCCTCT TGCCCGCTAAGTGTCGGTCACTCTGTCATAGGCCGCGCGCTGCTGCAGCACATCCAGTACCTGCTGA GCGTTAGCTTTCAGATCTTCATGCCCGTGTAAACGCATCAATATGGCGACGTTGGCGGCGACGGCGG CTTCGTGAGCGGCTTCACCTTTACCTTG MUT31

A Klebsiella bacterial mutant (MUT31) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding N-acetylglucosamine-6-phosphate deacetylase (VIR31). The insertion site nucleic acid sequence identifying the VIR31 gene in MUT31 is shown in Table 33. TABLE 33 MUT31 Transposon Insertion Site (SEQ ID NO: 49) TGGCTCAACGCTGCTCAGTGGTGCGAGGTGTCACTTTGGTGATCACATCGGCGTTGTCTGCACAGTG AAATCAGATCCAGCGCCGCGTCCGGTTTTACGCACGTAGTCCGGATTGTGGGTGCCTTTCTTAACGA TATTCAGCCACGGCCCTTCGAGATGCAGGCCCAGCGCCTGGTTCGGATGTTTTTGCAGATATTCGCG CATCACGCGCACGCCTTGCTTCATCAGATCGTCGCTGGAGGTAATCAGCGTCGGCAGGAAGCTGGTG CAGCCTGAGCGTTCGTTGGCCTTCTGCATGATCTCCAGCGTTTCGACAGTGACCGCCTCTGGGCTGT CGTTAAACTGCACGCCGCCGCAGCCGTTGAGCTGGACGTCGATAAAACCGGGGGCGATTATTGCGCC GTTGACTGAGCGCTGCTCGATGTCAGACGGCAAATCTGCCAGCGGACAAAGACGTTCGATAAAG MUT32

A Klebsiella bacterial mutant (MUT32) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding WaaQ (VIR32; Regué et al. J. Bacteriol. 183(12): 3564-73, 2001). The insertion site nucleic acid sequence identifying the VIR32 gene in MUT32 is shown in Table 34. TABLE 34 MUT32 Transposon Insertion Site (SEQ ID NO: 50) TTAAGCACCATATCGTACCGCTGCTGGCGCAGCGTCTGAATGAGCTGCCATTGCATCTTCAGCTGAT ACCTTTTTCCCTGGCTTTTTCCAGCGGCGATCGAGACCATAAATATGGTGGATATCGGGGTTGGCTG CGAGCATATCCCGGGTCTCTTCATACAACAGGACATCCACGCTGGCGGCGGGGTACTGCTGTTTCAG CGCGTGAATAAGCGGCGTGATCAGCAGCATGTCGCCATGATGGCGCAGCTTAATGACCAGGATCCGC GCCGGGTTCAACGGGCCGCGGGAGAGGGTTTCAGGCGTCATACTCTGTTCTTCATCCAGGATAAGGG TTCCGATTCTAGGGGATCAGACAGATTGAGAGAAGCGTTGTATTGCTCTACCATGACCCGATACGTA TGGCCTGAGGACGTTTTCGTGCACAATCCCGCAATTTTCTCATCACGAT MUT33

A Klebsiella bacterial mutant (MUT33) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding 2-isopropylmalate synthase (VIR33). The insertion site nucleic acid sequence identifying the VIR33 gene in MUT33 is shown in Table 35. TABLE 35 MUT33 Transposon Insertion Site (SEQ ID NO: 51) CACTCAGGCTTGCCTGTAACGCTTGTTCGCCATCACGTAAGGTCGTATCGAAAATAATGACTTGCTG GCTCATGGTTTGGATCCTTAGTCTGTGTCCTGGCGCCTTGTTGACGAGCATAAAAAAACCCGCGCCA AGGCGCGGGTTTTATAGTCTTGCTGGAAGATGACTTAACGCTGAACGTCGCCCAACAGCCTACCGAG CAAATGGCATGCGTTTAGTAGTAGTAGGCTGGTGATACGAGCGGTGCGAATCATTGCGTCAAACTCC AGATGAAATCGTTATGCTTTTAGAGTTACTGGATAGCCGTTTTAAAGTCAACCCCTGGCATGGAAAA AGCGTTTTGGGCTGACTAAATGAATTAGCAAAATGTGCTGATGTAAGCCCCATTTTGCCGAAGATCC TATTTTGGACCGAAGGCGGTTTATCCCCAATTTGTTTCATTTGAAAAA MUT34

A Klebsiella bacterial mutant (MUT34) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding histidinol dehydrogenase (VIR34). The insertion site nucleic acid sequence identifying the VIR34 gene in MUT34 is shown in Table 36. TABLE 36 MUT34 Transposon Insertion Site (SEQ ID NO: 52) CGCTGAACCGCTATCCGGAGCCGCAGCCGAAGTGCCGTGATTGAGAGCTACGCCCGCTACGCCGAGG TCAAACCGGAGCAGGTGCTGGTCAGCCGCGGCGCCGACGAAGGCATCGAGCTGCTGATCCGCGCCTT CTGTGAGCCCGGCGAAGACGCGGTGCTCTACTGCCCGCCGACCTACGGCATGTACAGCGTCAGCGCC GAGACCATCGGCGTCGAGTGCCGCACCGTGCCGACGCTGGCCAGCTGGCAGCTCGACCTGCCGGGCA TCGAAGCGCGGCTGGACGGCGTGAAGGTGGTGTTTGTCTGCAGCCCGAACAACCCGACCGGGCAGAT TATCGACCCGCAGTCGATGCGCGACCTGCTGGAGATGACCCGCGGCAAAGCCATCGTGGTGGCCGAC GAAGCCTATATTGAATTCTGCCCGCAGGCGACGCTCGCCGGCTGGCTCAGCGACTATCCGCACCTGG TGGTGCTGCGCACGCTGTCCAAAGCCTTCGCCCTCGCCGGCCTGCGCTGCGGCTTCACCCTCGCCAA CGCCGAGGTGATTAACGTGCTGCTGAAAGTGATCGCCCC MUT35

A Klebsiella bacterial mutant (MUT35) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding UDP-galactopyranose mutase (VIR35; Clarke et al., J. Bacteriol., 177: 5411-18, 1995). The insertion site nucleic acid sequence identifying the VIR35 gene in MUT35 is shown in Table 37. TABLE 37 MUT35 Transposon Insertion Site (SEQ ID NO: 53) CGTATATTTCATCGTACAGAAACCGTAAACACAGGCATTGGCTGATTTTCAGTGAGTGAATTTAAAT AGACTTCTGCCGTTTTCAATGCTTCGGCGATGGTCACATCCATATCAAGGTAACGGTAGGTTCCAAG ACGACCGACAAAAGTGATGTTGGTTTCATTCTCGGCCAATGACAAATATTTTTCAAGAAGAGCCATT TCTCCCATCTGGCGAATAGGATAGTAAGGAATATCATTTTCTTCACAAGCACGGCTATACTCTTTAT AACAAACAGAGCCGTCGTGTTGTTCCCAGGGAGAAAAATATTTATGTTCAGTGATGCGAGTATAGGG CACATCCACAGAACAGTAGTTCATCACTGCGCATCCCTGG MUT36

A Klebsiella bacterial mutant (MUT36) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the scene encoding O-antigen export system permease protein rfba (VIR36; Bronner et al., Mol. Microbiol., 14: 505-19, 1994). The insertion site nucleic acid sequence identifying the VIR36 gene in MUT36 is shown in Table 38. TABLE 38 MUT36 Transposon Insertion Site (SEQ ID NO: 54) GTACGCCGATTTTATATGCGTCTGATATGATTCCGGAAAAATTTAGCTGGATAATTACCTACAATCC GCTAGCGAGTATGATTCTTAGTTGGCGTGATTTATTCATGAATGGGACTCTTAATTTTGAGTATATT TCTATACTCTATTTTACGGGAATTATTTTGACGGTTGTCGGTTTGTCTATTTTCAATAAATTAAAAT ATCGATTTGCAGAGATCTAAAAGTGCGCTATAAGAGCAGCATGCTAGGCTATTTATGGTCAGTAGCA AATCCATTGCTTTTTGCCATGATTTACTATTTTATATTTAAGCTGGTAATGAGAGTACAAATTCCAA ATTATACAGTTTTCCTCATTACCGGCTTGTTTCCGTGGCAATGGTTTGCCAGTTCGGCCACTAAC MUT37

A Klebsiella bacterial mutant (MUT37) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding uridyltransferase (VIR37). The insertion site nucleic acid sequence identifying the VIR37 gene in MUT37 is shown in Table 39. TABLE 39 MUT37 Transposon Insertion Site (SEQ ID NO: 55) CGAGCCACCCACTGTAGCGTATGGATATCGCGCAAGCCGCCGGGGCTGCTTTTCACGTCCGGCTCGA GGTTATAGCTGGTGCCATGATAGCGCTGATGACGGACGTTCTGCTCTTCGACCTTGGCGGCGAAGAA CTTTTCCGATGGCCAGAAGCCGTCGCTAAAAATATGTTTTTGCAGTTCAAGGAACAGCGCGACGTCG CCGATCAGCAGGCGCGATTCGATTAAGTTGGTGGCAACGGTCAGATCCGAGAGACCTTCCAGCAGGC ACTCTTCGAGGGTGCGTACGCTGTGGCCCACCTCCAGCTTGACGTCCCACAGCAGGGTGAGCAGTTC GCCGACTTTTTGCGCCTGGTCGTCCGGCAGTTTTTTACGACTGAGGATCAGCAGATCGACGTCTGAG AGCGGGTGCAG MUT38

A Klebsiella bacterial mutant (MUT38) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding pyridoxine phosphate biosynthetic protein PdxJ-PdxA (VIR38). The insertion site nucleic acid sequence identifying the VIR38 gene in MUT38 is shown in Table 40. TABLE 40 MUT38 Transposon Insertion Site (SEQ ID NO: 56) CTTAACCCGCACGCTGGCGAAGGCGGCCATATGGGAACAGAAGAGATAGACACCATCATTCCGGTGC TGGAAGAGATGCGCGCAAAGGGGATGAACCTCAGCGGTCCGCTGCCGGCAGACACTCTCTTTCAGCC GAAATATCTTGATCATGCCGATGCGGTACTCGCGATGTACCACGATCAGGGCCTGCCCGTGCTAAAA TACCAGGGCTTTGGCCGCCGCGTGAACATTACGCTCGGTTTACCTTTTATTCGTACCTCCGTCGACC ACGGCACCGCACTGGAATTAGCGGGCCAGGGAAAAGCGGACGTCGGCAGTTTTATCACGGCGCTTAA TCTCGCCATCAAAATGATTGTTAATACCCAATGAATAATCGAGTCCATCAGGGCCATTTAGCCCGCA AACGCTTCGGGCAGAACTTCCTCAACGATCAGTTTGTGATCGACAGCATCGTCTCGGCGATTAACCC GCAGAAAGGCCAGGCGATCGTTGAAATCGGC MUT39

A Klebsiella bacterial mutant (MUT39) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding triose phosphate isomerase (VIR39). The insertion site nucleic acid sequence identifying the VIR39 gene in MUT39 is shown in Table 41. TABLE 41 MUT39 Transposon Insertion Site (SEQ ID NO: 57) GGGTCTGACCCCGGTTCTGTGCATCGGTGAAACCGAAGCCGAAAACGAAGCGGGCAAAACGGAAGAA GTTTGCGCACGTCAGATCGACGCCGTGCTGAAAACCCAGGGCGCTGCCGCTTTCGAAGGCGTGGTTA TCGCTTACGAACCAGTATGGGCTATCGGTACCGGCAAATCAGCGACCCCGGCTCAGGCGCAGGCGGT GCACAAATTCATCCGTGACCACATTGCTAAAGCTGACGCCAAAATCGCTGAGCAAGTGATCATCCAG TACGGCGGTTCCGTTAACGCTGGCAACGCCGCAGAGCTGTTCACCCAGCCGGACATCGACGGCGCGC TGGTTGGCGGCGCCTCCCTGAAAGCTGACGCTTTCGCGGTGATCGTTAAAGCAGCAGAAGCAGCGAA AAAAGCGTAATTCGCTTTTCCCGGTGGCGACACGCGACCGGGTTGACTGACAAAACGTGGGAGCCCG GCCT MUT40

A Klebsiella bacterial mutant (MUT40) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding aldehyde dehydrogenase (VIR40). The insertion site nucleic acid sequence identifying the VIR40 gene in MUT40 is shown in Table 42. TABLE 42 MUT40 Transposon Insertion Site (SEQ ID NO: 58) GGTGGCGCACCCTGGCGTCGTTTGTGTAGAAATTATGAATATTAATACCAGGAAAATTCCTAATTTT TGTGTACGCTCTGACGAGCGCACAATAAAACAAGACGAATTTTTGAACAATTGTCTTTAAATTTGTT AATTGAATTGATCTGTTGTTGTTTAAAGGTATTTGAATTTCTTTTGTATAGATATGTAAATTAACAT TGAAAAGCCATTTCAAAAATTAAATATATGGCGAACATAGCTATTAACTTATAGTTAACATCTTCCC GGGTTGCCTTTTGATACTTCGGGTAATATATTTATTTCGCACATCAAAATAACTCTTTTTTCTTCTG TTTGTTATTCATGGCCATCTATTGGCGAAATAAGGCAGAGTAGAGGGGGATGTGCCTAATATCCTGC GGAAGGAACGCAATGTACATTTACAGGGAGGAGCTGACGAGCCGTTTCGCGATAGCTTTAG MUT41

A Klebsiella bacterial mutant (MUT41) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding galacosyl transferase (VIR41; Clarke et al., J. Bacteriol., 177: 5411-18, 1995). The insertion site nucleic acid sequence identifying the VIR41 gene in MUT41 is shown in Table 43. TABLE 43 MUT41 Transposon Insertion Site (SEQ ID NO: 59) TTGGTGGTGTGCTCGCGAAGAAATTTAATCTGCCGGTCATCGTAAGTTTTGTTGGGCTTGGAAGAGT ATTTTCTTCTGACAGCATGCCTTTAAAATTATTGCGGCAGTTTACTATTGCTGCATATAAATATATT GCCAGTAATAAGCGCTGTATATTTATGTTTGAACATGACCGCGACAGAAAAAAACTGGCTAAGTTGG TTGGACTCGAAGAACAACAGACTATTGTTATTGATGGTGCAGGCATTAATCCAGAGATATACAAATA TTCTCTTGAACAGGATCACGATGTCCCTGTTGTATTGTTTGCCAGCCGTATGTTGTGGAGTAAAGGA CTGGGCGACTTAATTGAAGCGAAGAAAATATTACGCAGTAAGAATATTCACTTTACTTTGAATGTTG CTGGAATTCTGGTCGAAAATGATAAAGATGCAATTTCCCTTCAGGGTCATTGAAAATTGGCATCAGC AAGGATTAATTAACTGGTTAGGTCGTTCGAATAATGTTTGCGATCTTATTGAGCAAT MUT42

A Klebsiella bacterial mutant (MUT42) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding siroheme synthetase (VIR42; Kolko et al., J. Bacteriol., 183: 328-35, 2001). The insertion site nucleic acid sequence identifying the VIR42 gene in MUT42 is shown in Table 44. TABLE 44 MUT42 Transposon Insertion Site (SEQ ID NO: 60) TTACTTGCCCCTTTTTGCCGAACTGAAACAAAGGCCCGTGCTGGTGATCGGCGGCGGCGAGATTGCT GAACGTAAGATCAAGTTCCTGCTGCGCGCCCAGGCGCAGGTGCAGGTGGTCGCTGAAACGCTGTCAC CGGCGCTGGCCGATCTGGCTGCGCGCCAGGCACTCAGCTGGCGGGCGACGGCATTCAGCGACTCGCT GGTGGATGATGTCTTTCTGGTGATTGCGGCCACCGAGGATGAGGCGCTTAACCAGCGGGTGTTTGCG GCAGCTAACGCGCGCTACCGGTTGGTCAACGTGGTGGATAACCAGGCGCTGTGCTCGTTTGTTTTCC CTTCTATCGTCGACCGTTCGCCGCTGCTGGTGGCGATCTCCTCCAGCGGTAAAGCGCCGGTGTTGTC GCGCATTCTGCGTGAAAAAATCGAAGCGCTGCTGCCGACGAATCTCGGTCGGCTGGCGCAATCAGCA AGCT MUT43

A Klebsiella bacterial mutant (MUT43) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (VIR43). The insertion site nucleic acid sequence identifying the VIR43 gene in MUT43 is shown in Table 45. TABLE 45 MUT43 Transposon Insertion Site (SEQ ID NO: 61) AGCAGGGCAATGGTGGTCGGTTTCATAACATTTCCTGATGATGAAAGTCATATTAACCGCCATTCTA ACAGCAGCATTCAGAGGGGCAATGATTTTGGGCAACCGATTACGACGATCGCCGCAAATGCTAAAAA AGGGAGAGGGGATTACCAGCTGGCGGGCTTTTCCGCGCCGAGATTATCCAGCACGGCGCGCAGCGCC AGGCCGTCAGGAAAGTGAAGGTCCGGGGCGATCTCGAACAGCGGCCAGAGCATAAAGCCGCGGTTTT TCATATCGTAGTGCGGAACGGTCAGGCGCTCGCTGTTAATGACAGCATCGCCAAACAGCATGATATC GAGGTCCAGCGTGCGCGGCCCCCAGCGTTCGGCTTTGCGCACTCGCCCCTGCTGCAGTTCGATGCGC TGAGTATGATCGAGCAGCGTCTCGGGGGGCAGGGCGGTTTCCAGCGCAA MUT44

A Klebsiella bacterial mutant (MUT44) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding glucose-6-phosphate isomerase (VIR44). The insertion site nucleic acid sequence identifying the VIR44 gene in MUT44 is shown in Table 46. TABLE 46 MUT44 Transposon Insertion Site (SEQ ID NO: 62) GGCTTAACGCCAGCTATGTCAACGCTGCGGTTATGCGGATTTTTCATGCCTCTGCGGCTAACAGAAA AAAGCCTTATGATAGCTATACTAATGGGGCTTTTTACTCCGTTTTGACCCGATTCCTGACCGGCGTC AGGGTCAAGTCACAAAAATCATCACAATTTTCCGTCACCGGCGCTACAATCGACCGAAGTCACAATC TCAAATCAGAAGAGTATTGCTAATGAAAAACATCAACCCAACGCAGACCTCTGCCTGGCAGGCATTA CAGAAACACTTCGACGAAATGAAAGATGTCACTATCAGCGAGCTTTTCGCCAAAGATAGCGACCGTT TTTCTAAATTTTCCGCGACGTTCGACGATCTGATGCTGGTGGACTTCTCCAAAAACCGCATCACTGA AGAGACGCTGGCTAAACTGCAGGATCTGGCGAAAGAGACTGACCTGGCGGGCGCTATCAAGTCGATG TTCTCAGGTGAGAAGATCAACCGCACCGAAGACCGCGCGGTACTGCACGTCGCGCT MUT45

A Klebsiella bacterial mutant (MUT45) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding DNA methylase (VIR45). The insertion site nucleic acid sequence identifying the VIR45 gene in MUT45 is shown in Table 47. TABLE 47 MUT45 Transposon Insertion Site (SEQ ID NO: 63) TGCTTCATCCGCATCTCCTTGAAATTTATTTGGTCTTAGGCGGACGGTAGAGCGCTAATAGCTCGTC CACCTTTTTACGCGTACCACCGTTGCTGCTGATGCTGCGCCGCACCTTCACAATATGCGTTTCTGCC GCGTTTTTATACCATTCCTGCGTCAGCGGCGTGCGGTGGTTGGAAATCAGCACCGGGATGCGCTTTT TCATCAGCGATTCCGCCTTTTGCGCCAGCAGTACCTGTTGTTCCAGGTTGAAACTGTTGGTGTGGTA GGCGGTAAAGTTCGCCGTCGCCGTTAGCGGCGCATAGGGCGGATCGCAATACACCACTGTGCGGCTA TCCGCACGTTGCATGCACTCTTCGTAAGATTCGCAGTAAAACTCGGCGTTTTGCGCCTTCTCGGCGA AATGATAGAGCTCAGCTTCGGGGAAATAGGGCTTTTTATAACGGCCAAACGGCACATTGAACTCGCC GCGCAG MUT46

A Klebsiella bacterial mutant (MUT46) was made by transposon insertion in a Klebsiella sp. wild-type strain. In the Dictyostelium growth assay, the mutated microorganism was less virulent compared to an isogenic bacterial strain. The nucleotide sequence immediately following the transposon insertion was cloned and identified as the gene encoding a putative inner membrane protein (VIR46). The insertion site nucleic acid sequence identifying the VIR46 gene in MUT46 is shown in Table 48. TABLE 48 MUT46 Transposon Insertion Site (SEQ ID NO: 64) TGTCAATGCGCAATTTGGTTAAATATGTCGGTATTGGCCTGCTGGTGATGGGGCTTGCCGCCTGCGA TAACAGCGATTCAAAAGCGCCAACCGTTGGCGCAGCAGCGGAGAGCAATGCCAGCGGCCAGGCAATC AGCCTGCTGGATGGCAAGCTGAGCTTCACCCTGCCTGCGGGCATGGCCGACCAGAGCGGCAAACTGG GTACCCAGGCGAACAATATGCACGTCTACTCTGACGCTACCGGCCAGAAAGCGGTCATCGTCATCGT CGGCGACAGCACCAATGA IV. Suitable Target Pathogens

Other Pseudomonas sp. and Klebsiella sp. and many other microbes, including gram-negative bacterial strains, are likely to include virulence genes encoding VIRX-related peptides or proteins having amino acid sequence identity or similarity to those identified herein. Suitable bacterial pathogens may include, but are not limited to, Pneumococci sp., Klebsiella, sp., Pseudomonas, e.g., P. aeruginosa, Salmonella, e.g., Salmonella typhimurium, Legionella, e.g., Legionella pneumophilia, Escherichia, e.g., Escherichia coli, Listeria, e.g., Listeria monocytogenes, Staphylococcus, e.g., Staphylococcus aureus, Streptococci sp., Vibrio, e.g., Vibrio cholerae. Pathogenic mycobacteria of the present invention may include e.g., Mycobacterium tuberculosis. Pathogenic fungi of the present invention may include, e.g., Candida albicans. Pathogenic unicellular eukaryotic organisms of the present invention may include, e.g., Leishmania donovani.

Having identified VIRX genes according to the invention, it is possible to use the gene sequence to search for related genes or peptides in other microorganisms. This may be carried out by searching in existing databases, e.g., EMBL or GenBank. The levels of identity between gene sequences and levels of identity or similarity between, amino acid sequences can be calculated using known methods. In relation to the present invention, publicly available computer based methods for determining identity and similarity include the BLASTP, BLASTN and FASTA (Atschul et al., J. Molec. Biol., 1990; 215:403-410), the BLASTX program available from NCBI, and the Gap program from Genetics Computer Group, Madison Wis.

Preferably, the peptides that may be useful in the various aspects of the invention have greater than a 40% similarity with the peptides identified herein. More preferably, the peptides have greater than 60% sequence similarity. Most preferably, the peptides have greater than 80% sequence similarity, e.g., 95% similarity. With regard to the polynucleotide sequences identified herein, related polynucleotides that may be useful in the various aspects of the invention may have greater than 40% identity with the sequences identified herein. More preferably, the polynucleotide sequences have greater than 60% sequence identity. Most preferably, the polynucleotide sequences have greater than 80% sequence identity, e.g., 95% identity.

In addition to related molecules from other microorganisms, the invention encompasses modifications made to the peptides and polynucleotides identified herein which do not significantly alter the biological function. It will be apparent to the artisan that the degeneracy of the genetic code can result in polynucleotides with minor base changes from those specified herein, but which nevertheless encode the same peptides. Complementary polynucleotides are also within the invention. Conservative replacements at the amino acid level are also envisaged, i.e., different acidic or basic amino acids may be substituted without substantial loss of function.

It is recognized in the art that highly refined mechanisms that regulate transcription have evolved and are present in bacteria. Most bacterial genes are organized into operons, which are groups of genes coding for related proteins. Operons can either be repressed or induced thus regulating those genes. An operon consists of an operator, promoter, regulator, and structural genes. The regulator gene codes for a repressor protein that binds to the operator, obstructing the promoter (thus, transcription) of the structural genes. The regulator does not have to be adjacent to other genes in the operon. If the repressor protein is removed, transcription may occur.

Transposon mutagenesis usually inactivates the gene in which the transposon is inserted, as well as any gene downstream in the same operon. If the VIRX gene is a structural gene in an operon, inactivation of the MAX gene disrupts the expression of other structural genes in the same operon and positioned downstream of the inactivated VIRX gene. For example, an insertion in pchE gene also inactivates pchF, pchG, pchH, and pchI genes because they all reside within the pchEFGHI operon and are downstream of the inactivated pchE gene. Accordingly, the present invention includes attenuation of virulence due to alteration of a VIRX gene residing in an operon as well as alterations to nucleic acid yielding loss of expression of structural genes located in the same operon and located downstream of the VIRX gene. In one embodiment, the present invention is an alteration inactivating the first gene of an operon carrying a VIRX gene of the invention. The alteration of nucleic acids of VIRX genes and VIRX-containing operons may be insertional inactivation or gene deletion. It is preferred that the alteration of nucleic acids of VIRX genes and VIRX-containing operons be insertional inactivation.

The present invention also provides for a bacterial strain comprising an operon encoding a gene selected from the group consisting of VIR1, VIR2, VIR3, VIR4. VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14, VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR44, VIR45, and VIR46, wherein the bacterial strain includes a mutation that reduces expression of the VIRX gene relative to an isogenic bacterial strain lacking the mutation. In one embodiment, the mutation reduces inhibition of Dictyostelium amoeba growth when compared to the growth of Dictyostelium amoeba in the presence of an isogenic bacterial strain lacking the mutation. In another embodiment, the attenuated bacterial strain has more than one mutation of an operon containing a VIRX gene when compared to an isogenic bacterial strain.

V. VIRX Nucleic Acids and Polypeptides Can Be Used to Identify Antimicrobial Drugs

A. Screening

In a separate embodiment, the VIRX genes, or their polynucleotide or polypeptide products disclosed herein is used in screening assays for the identification of potential antimicrobial drugs. Routine screening assays are known to those skilled in the art, and can be adapted using the VIRX products of the invention in the appropriate way. For example, the products of the invention can be used as the target for a potential drug, with the ability of the drug to inactivate or bind to the tar-get indicating its potential antimicrobial activity. In the methods of the present invention, one or more test compounds may be present or produced in the assay mixture. Preferably one compound is present, or produced, in the assay mixture.

B. Character of Antimicrobial Candidate Compositions

VIRX nucleic acids and polypeptides may be used to identify drugs or therapeutics in a candidate composition useful in the prevention or treatment of pathogen-associated disease or infection. A candidate composition can include one or more molecules for analysis in a screening assay and can be a synthetic or semi-synthetic molecules. Such molecules include inorganic as well as organic chemical molecules. The molecules may be less than about 500 Daltons or more than 500 Daltons. The molecules may be naturally occurring. Naturally occurring molecules may include, e.g., saccharides, lipids, peptides, proteins, nucleic acids, or combinations thereof, e.g., aminoglycosides, glycolipids, lipopolysaccharides, or macrolides. Proteins may be immunoglobulins, e.g., polyclonal or monoclonal antibodies. Nucleic acids may be DNA or RNA, e.g., small interfering RNA (siRNA). The precise source of the molecule is not critical to the method of the present invention. The molecule might be derived from e.g., synthetic compounds libraries that are commercially available, e.g., Sigma-Aldrich (Milwaukee, Wis.), or libraries of natural occurring molecules in the form of bacterial, fungal, plant, and animal extracts such as those available from Xenova (Slough, UK). The synthetic (or semi-synthetic) or natural occurring molecules might be modified using standard chemical, physical, or biochemical methods known in the art.

VI. VIRX Nucleic Acids and Polypeptides Can Be Used to Detect the Degree of Virulence of Pathogens

A diagnostic test can assist physicians in determining the type of disease and appropriate associated therapy. As such, a separate embodiment of this invention provides for the use of VIRX genes or their polynucleotides or nucleic acid products as virulence markers for detecting the presence of a pathogen, a pathogen-associated disease, or the virulence of a pathogen. There are many diagnostic assay approaches known to the artisan. Generally, the diagnostic method used would comprise the steps of (a) obtaining a sample from a potentially diseased subject or a diseased subject; (b) measuring the level of at least one polypeptide or polynucleotide virulence marker in the sample; and (c) comparing the amount of the virulence marker in the sample of step (a) to the amount of the virulence marker present in a control sample from a second subject known not to have the presence of the pathogen, where an alteration in the expression level of the virulence marker in the first subject as compared to the control sample indicates the presence of a pathogen, a pathogen-associated disease, or the virulence of a pathogen. Preferably, the subject is a mammal. More preferred is that the subject is a human. The person of skill will recognize that diagnostic tests may be performed in an array-type format wherein, e.g., the presence of two or more VIRX genes or gene products indicate the presence of a pathogen, a pathogen-associated disease, or the virulence of a pathogen.

VII. Attenuated Organisms of the Present Invention Can Be Used in Vaccine Preparation

In another embodiment, the invention provides for the use of the attenuated organisms described herein in vaccine preparation. The preparation of vaccines based on attenuated microorganisms is known to those skilled in the art. Vaccine compositions can be formulated with suitable carriers or adjuvants, e.g., alum, as necessary or desired, to provide effective immunization against infection. The preparation of vaccine formulations will be apparent to the artisan. The attenuated microorganisms may be prepared with a mutation that disrupts the expression of any of the VIRX genes identified herein. The artisan will be aware of methods for disrupting expression of particular VIRX genes. Techniques that may be used include, but are not limited to, insertional inactivation, or gene deletion techniques. Attenuated microorganisms according to the invention may also comprise additional mutations in other genes, for example in a second gene identified herein or in a separate gene required for growth of the microorganism, e.g., an Aro mutation. Attenuated microorganisms may also be used as carrier systems for the delivery of heterologous antigens, therapeutic proteins or nucleic acids (DNA or RNA). In this embodiment, the attenuated microorganisms are used to deliver a heterologous antigen, protein or nucleic acid to a particular site in vivo. Introduction of a heterologous antigen, peptide or nucleic acid into an attenuated microorganism can be carried out by conventional techniques, including the use of recombinant constructs, e.g., vectors, which comprise polynucleotides that express the heterologous antigen or therapeutic protein, and also include suitable promoter sequences. Alternatively, the gene that encodes the heterologous antigen or protein may be incorporated into the genome of the organism and the endogenous promoters used to control expression. In the vaccines of the present invention, the pharmaceutically effective dosage of the mutants of the present invention to be administered may vary depending on the age, weight and sex of the subject, and the mode of administration. The subject can be, e.g., a human, a non-human primate (such as an ape, gorilla, or chimpanzee), cow, horse, pig, sheep, dog, cat, or rodent (including mouse or rat).

VIII. Definitions

As used herein, each of the following terms has the meaning associated with it in this section.

The term “pathogen,” as used herein, is intended to include an agent that causes disease, especially a living microorganism such as a bacterium or fungus. The terms “agent” and “factor” are used interchangeably herein to describe pathogens or toxins useful in the methods of the present invention. Pathogens may include any bacteria, mycobacteria, fungi and unicellular eukaryotic organism, including wild types and mutants thereof, which causes disease or brings about damage or harm to a host organism. Pathogens may also be a poisonous substance, e.g., toxin, which is produced by living cells or organisms and is capable of causing disease when introduced to a host.

The term, “pathogenic,” as used herein, is defined as an agent's ability to cause disease, damage or harm to a host organism.

The term, “attenuated,” as used herein, means an organism made less virulent relative to an isogenic pathogenic organism.

The term, “virulence,” as used herein, is a measure of the degree of pathogenicity of an agent to a host organism. Virulence is usually expressed as the dose of an agent or cell number of a pathogen that will elicit a pathological response in the host organism within a given time period. “Reducing the virulence” as used herein is defined as the ability of a compound to attenuate, diminish, decrease, suppress, or arrest the development of, or the progression of disease, damage or harm to a host organism mediated by a pathogen.

The term, “host organism,” as used herein, is intended to include any living organism. Preferably the host organism is a eukaryote, e.g., vertebrate. More preferably the host organism is a mammal. It is most preferred that the host organism be a human.

The term, “mutant,” as used herein, an organism carrying a specific mutation of a gene that is expressed in the organism's phenotype.

The term, “mutation,” as used herein, is an alteration of one or more nucleic acids of a polynucleotide sequence encoding a gene. A mutation may include the insertion of additional nucleic acids to a polynucleotide sequence encoding a gene, e.g., insertional inactivation of a gene. Alternatively, a mutation may include, but is not limited to, deletion of one or more nulceic acids of a polynucleotide sequence encoding a gene.

The term, “operon,” as used herein, is a unit of bacterial gene expression and regulation comprising several genes usually with complementary functions. Typically an operon includes nucleic acid and control elements in the nucleic acid that may be recognized by regulators of gene products. Insertion in a gene in an operon interferes with the function of this gene and of other genes located downstream or upstream in the operon. It is understood herein that the function attributed to a gene refers to its function and/or that of any gene located downstream or upstream in the same operon.

The term, “pharmaceutically effective dosage,” as used herein, means that amount necessary at least partly to attain the desired effect, or to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the particular condition being treated.

The terms “similarity” and “identity” are known in the art. The use of the term “identity” refers to a sequence comparison based on identical matches between correspondingly identical positions in the sequences being compared. The term “similarity” refers to a comparison between amino acid sequences, and takes into account not only identical amino acids in corresponding positions, but also functionally similar amino acids in corresponding positions. Thus similarity between polypeptide sequences indicates functional similarity, in addition to sequence similarity.

EQUIVALENTS

From the foregoing detailed description of the specific embodiments of the invention, it should be apparent that bacterial genes have been identified and assigned a new role in virulence. Further, these genes and their products are useful in the identification of antimicrobial agents, the diagnosis of pathogen-associated disease or infection as well as the preparation of vaccines. Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims that follow. In particular, it is contemplated by the inventor that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. For instance, the choice of the particular pathogen, or combination of pathogens selected for assay or vaccination, the test conditions used in diagnostic assays utilizing the pathogens of this invention, or the method of mutagenesis used to derive the attenuated mutants is believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein.

EXAMPLES

This Example is provided for the purpose of illustration only and the invention should in no way be construed as being limited to these Example, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided.

Example 1 Strains and Culture Conditions Used to Screen for Attenuated Viurlence in Test Bacterial Mutants

The D. discoideum wild-type strain DH1-10 used in these studies is a subclone of DH1 (Cornillon et al., J. Biol. Chem., 275(44):34287-92, 2000). Cells were grown at 21° C. in HL5 medium (14.3 g/l peptone (Oxoid), 7.15 g/l yeast extract, 18 g/l maltose, 0.64 g/l Na₂HPO₄.2H₂O, 0.49 g/l KH₂PO₄, pH 6.7) (Cornillon et al., J. Cell. Sci., 107 (Pt 10):2691-704, 1994) and subcultured twice a week.

Bacteria were grown overnight at 37° C. on Luria-Bertani (LB) agar. Single colonies were inoculated into 5 ml PB (2% (wt/vol) peptone, 0.3% (wt/vol) MgCl₂.6H₂O, 1% (wt/vol) K₂SO₄) (Essar et al., J. Bacteriol., 172(2):884-900,1990) in a 50 ml flask and grown at 37° C. for 8 hr prior to use. The growth of various strains was tested in rich medium (PB) by measuring the optical density (600 nm) of a culture at different times after inoculation and was found to be comparable for all strains used. Under these conditions, similar OD_(600s) were obtained for each strain and the induction of quorum sensing was maximal. Minimal Inhibitory Concentrations (MICs) were determined in Mueller-Hinton broth by the microdilution method (Thornsberry et al., NCCLS, 3: 48-56, 1983). Mutations yielding reduced virulence were identified where the growth of the Dictyostelium test host organism exposed to the mutant pathogen was greater than the Dictyostelium test host organism exposed to wild-type pathogen. Specific genetic mutations in pathogens displaying reduced virulence were identified and characterized by techniques well know in the art. 

1. An attenuated bacterial mutant derived from a pathogenic bacterial strain, wherein said attenuated mutant has: (i) a mutation of a gene selected from the group consisting of VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14. VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR43, VIR44, VIR45, and VIR46; and (ii) reduced inhibition of Dictyostelium amoeba growth when compared to the growth observed in the presence of an isogenic bacterial strain.
 2. An attenuated bacterial mutant of claim 1, wherein said mutation is insertional inactivation or a gene deletion.
 3. An attenuated bacterial mutant of claim 1, wherein said mutant is a gram-negative bacteria.
 4. An attenuated bacterial mutant of claim 3, wherein said attenuated gram-negative bacterial mutant is a Pseudomonas species.
 5. An attenuated bacterial mutant of claim 4, wherein said Pseudomonas species is Pseudomonas aeruginosa.
 6. An attenuated Pseudomonas mutant of claim 5, wherein said attenuated Pseudomonas mutant is selected from the group consisting of: MUT1; MUT2; MUT3; MUT4; MUT5; MUT6; MUT7; MUT8; MUT9; MUT10; MUT11; MUT12; MUT13; MUT14; MUT15; MUT16; MUT17; MUT18; and MUT19.
 7. An attenuated bacterial mutant of claim 3, wherein said gram-negative bacterial mutant is a Klebsiella species.
 8. An attenuated bacterial mutant of claim 7, wherein said Klebsiella species is Klebsiella pneumoniae.
 9. An attenuated Klebsiella mutant of claim 8, wherein said attenuated Klebsiella mutant is selected from the group consisting of: MUT20; MUT21; MUT22; MUT23; MUT24; MUT25; MUT26; MUT27; MUT28; MUT29; MUT30; MUT31; MUT32; MUT33; MUT34; MUT35; MUT36; MUT37; MUT38; MUT39; MUT40; MUT41; MUT42; MUT43; MUT44; MUT45; and MUT46.
 10. A method for identifying an antimicrobial drug, said method comprising: (a) contacting a candidate composition with at least one polypeptide encoded by a gene selected from the group consisting of VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14, VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR43, VIR44, VIR45 and VIR46; and (b) comparing the biological activity of said polypeptide in the presence and absence of said candidate composition, wherein alteration of the biological activity of said polypeptide indicates that said candidate composition is an antimicrobial drug.
 11. A method of claim 10, wherein said candidate composition contains at least two molecules.
 12. A method of claim 10, wherein said candidate composition contains at least one-molecule less than about 500 Daltons.
 13. A method of claim 10, wherein said candidate composition contains at least one molecule greater than about 500 Daltons.
 14. A method of claim 10, wherein said candidate composition contains at least one molecule selected from a group consisting of a polypeptide, polysaccharide, lipid, nucleic acid, or combination thereof.
 15. A composition of claim 14, wherein said polypeptide is an immunoglobulin.
 16. A method for identifying an antimicrobial drug, said method comprising: (a) contacting at a candidate composition with at least one polynucleotide encoded by a gene selected from the group consisting of VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14, VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR43, VIR44, VIR45, and VIR46; and (b) comparing the expression of said polynucleotide in the presence and absence of said candidate composition, wherein alteration of the expression of said nucleotide indicates that said candidate composition is an antimicrobial drug.
 17. A method of claim 16, wherein said candidate composition contains at least two molecules.
 18. A method of claim 16, wherein said candidate composition contains at least one molecule less than about 500 Daltons.
 19. A method of claim 16, wherein said candidate composition contains at least one molecule greater than about 500 Daltons.
 20. A method of claim 16, wherein said candidate composition contains at least one molecule selected from a group consisting of a polypeptide, polysaccharide, lipid, nucleic acid, or combination thereof.
 21. A composition of claim 20, wherein said nucleic acid is a ribonucleic acid.
 22. A nucleic acid of claim 21, wherein said nucleic acid is a small interfering ribonucleic acid.
 23. A method for determining the degree of virulence of a pathogen in a subject, said method comprising: (a) measuring the level of expression of at least one polypeptide encoded by a gene selected from the group consisting of VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14, VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR43, VIR44, VIR45, and VIR46, in a sample from the first subject; and (b) comparing the amount of said polypeptide in said sample of step (a) to the amount of said polypeptide present in a control sample from a second subject known not to have the presence of said pathogen, wherein an alteration in the expression level of said polypeptide in said first subject as compared to said control sample indicates the degree of virulence of said pathogen.
 24. A method of claim 23, wherein said subject is a mammal.
 25. A mammalian subject of claim 24, wherein said mammalian subject is a human.
 26. A method for determining the degree of virulence of a pathogen in a subject, said method comprising: (a) measuring the level of expression of at least one polynucleotide encoded by a gene selected from the group consisting of VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14, VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR44, VIR45, and VIR46, in a sample from the first subject; and (b) comparing the amount of said polynucleotide in said sample of step (a) to the amount of said polynucleotide present in a control sample from a second subject known not to have the presence of said pathogen, wherein an alteration in the expression level of said polynucleotide in said first subject as compared to said control sample indicates the degree of virulence of said pathogen.
 27. A method of claim 26, wherein said subject is a mammal.
 28. A mammalian subject of claim 27, wherein said mammalian subject is a human.
 29. An attenuated bacterial mutant of claim 1, wherein said mutant encodes and expresses a foreign antigen.
 30. An attenuated bacterial mutant of claim 1, wherein said mutant contains a plasmid which encodes and expresses, in a eukaryotic cell, a foreign antigen.
 31. A vaccine against a disease caused by a pathogenic microorganism comprising: (a) a pharmaceutically effective dosage of one or more of the attenuated bacterial mutants of claim 1 and; (b) a pharmaceutically acceptable diluent or carrier.
 32. An attenuated bacterial mutant derived from a pathogenic bacterial strain, wherein said attenuated mutant has: (i) a mutation of a gene selected from the group consisting of pchE, pchF, pchG, pchH, and pchI; and (ii) reduced inhibition of Dictyostelium amoeba growth when compared to the growth observed in the presence of an isogenic bacterial strain.
 33. A bacterial strain comprising an operon encoding a gene selected from the group consisting of VIR1, VIR2, VIR3, VIR4, VIR5, VIR6, VIR7, VIR8, VIR9, VIR10, VIR11, VIR12, VIR13, VIR14, VIR15, VIR16, VIR17, VIR18, VIR19, VIR20, VIR21, VIR22, VIR23, VIR24, VIR25, VIR26, VIR27, VIR28, VIR29, VIR30, VIR31, VIR32, VIR33, VIR34, VIR35, VIR36, VIR37, VIR38, VIR39, VIR40, VIR41, VIR42, VIR44, VIR45, and VIR46, wherein said bacterial strain includes a mutation that reduces expression of said gene relative to an isogenic bacterial strain lacking said mutation.
 34. A bacterial strain of claim 33, wherein said mutation reduces inhibition of Dictyostelium amoeba growth when compared to the growth of Dictyostelium amoeba in the presence of an isogenic bacterial strain lacking said mutation. 